Methods and compositions relating to microbial treatment and diagnosis of disorders

ABSTRACT

The present disclosure provides methods, systems, compositions, and kits to address the need for microbiome-related treatment of health conditions and disease. The present disclosure provides for treatment of metabolic conditions using microbial compositions.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.15/271,672, filed Sep. 21, 2016, which is a continuation of U.S.application Ser. No. 15/139,097, filed Apr. 26, 2016, now U.S. Pat. No.9,486,487, which is a continuation of PCT Application No.PCT/US15/58511, filed Oct. 30, 2015, which claims the benefit of U.S.Provisional Application Ser. No. 62/073,912, filed Oct. 31, 2014, all ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The body of an individual is inhabited by trillions of microbes acrossvarious locations, often referred to as microbiomes. Microbiomes canplay a key role in many health conditions and diseases. Despite theinterrelation between microbiomes and health, the complexity of thevarious microbiomes, as well as difficulties in characterizing,categorizing, and analyzing microbiome constituents has madeunderstanding microbiomes challenging. Consequently, these challengeshave presented hurdles in the development of diagnostic and therapeuticapplications for microbiome-related health conditions and diseases. Thepresent disclosure provides methods, systems, compositions, and kits toaddress the need for microbiome-related treatment of health conditionsand disease.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 21, 2016, isnamed 46790-702.302.txt and is 36,254,267 bytes in size.

BIOLOGICAL DEPOSITS

This application contains a reference to a deposit of biologicalmaterial. The following biological materials have been deposited withthe American Type Culture Collection (ATCC), in Manassas, Va., and bearthe following designations, accession numbers and dates of deposit:Clostridium beijerinckii; WB-STR-0005 (PTA-123634, deposited Dec. 14,2016); Clostridium butyricum; WB-STR-0006 (PTA-123635, deposited Dec.14, 2016).

SUMMARY OF THE INVENTION

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe that altersglucagon-like peptide-1 (GLP-1) production, and apharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe that encodesfor an enzyme selected from the group consisting of: butyrate kinase,butyrate coenzyme A, butyrate coenzyme a transferase, and anycombination thereof, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe that iscapable of producing butyrate, and a pharmaceutically-acceptablecarrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Akkermansiamuciniphila, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Anaerostipes caccae,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Bifidobacteriumadolescentis, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Bifidobacteriumbifidum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Bifidobacteriuminfantis, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Bifidobacteriumlongum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Butyrivibriofibrisolvens, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridiumacetobutylicum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridiumbeijerinckii, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridium butyricum,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridium colinum,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridium indolis,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Enterococcus faecium,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Eubacterium hallii,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Eubacterium rectale,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Faecalibacteriumprausnitzii, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Fibrobactersuccinogenes, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillusacidophilus, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillus brevis,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillusbulgaricus, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillus casei,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacilluscaucasicus, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillusfermentum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillushelveticus, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillus lactis,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillusplantarum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillus reuteri,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Lactobacillusrhamnosus, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Roseburia cecicola,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Roseburiainulinivorans, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Ruminococcusflavefaciens, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Ruminococcus gnavus,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Streptococcuscremoris, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Streptococcus faecium,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Streptococcusinfantis, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Streptococcus mutans,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Streptococcusthermophilus, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridiumaminophilum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridiumorbiscindens, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Oscillospiraguilliermondii, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Ruminococcus obeum,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Anaerofustisstercorihominis, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Anaerostipes hadrus,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Anaerotruncuscolihominis, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridiumsporogenes, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Clostridium tetani,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Coprococcus, and apharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Coprococcus eutactus,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Eubacteriumcylindroides, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Eubacterium dolichum,and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Eubacteriumventriosum, and a pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Roseburia faeccis, anda pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Roseburia hominis, anda pharmaceutically-acceptable carrier.

In some embodiments, this invention comprises a method of treating ametabolic disorder in a subject in need thereof, the method comprising:administering a therapeutically-effective amount of a pharmaceuticalcomposition comprising a population of isolated and purified microbe,wherein at least one of said microbes comprises a microbe with at leastabout 85% sequence identity to a rRNA sequence of Roseburiaintestinalis, and a pharmaceutically-acceptable carrier.

A method of treating a metabolic disorder in a subject in need thereof,the method comprising: administering a therapeutically-effective amountof a pharmaceutical composition comprising a population of isolated andpurified microbe, wherein at least one of said microbes comprises amicrobe with at least about 85% sequence identity to a rRNA sequence ofa microbe selected from the group consisting of: Akkermansiamuciniphila, Anaerostipes caccae, Bifidobacterium adolescentis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum,Clostridium aminophilum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium colinum, Clostridium indolis, Clostridiumorbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacteriumrectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes,Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillusbulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospiraguilliermondii, Roseburia cecicola, Roseburia inulinivorans,Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum,Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis,Streptococcus mutans, Streptococcus thermophilus, Anaerofustisstercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis,Clostridium sporogenes, Clostridium tetani, Coprocoaccus, Coprococcuseutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacteriumventriosum, Roseburia faeccis, Roseburia hominis, Roseburiaintestinalis, and any combination thereof.

The method of any of the preceding embodiments, wherein said treatingresults in a subject with an altered microbiome.

The method of any of the preceding embodiments, wherein said treatingresults in a subject with an altered gut microbiome.

The method of any of the preceding embodiments, wherein thepharmaceutical composition further comprises a second population ofisolated and purified microbe. In some aspects, the method may furthercomprise a second population of isolated and purified microbe, whereinsaid second population comprises a microbe with at least about 85%sequence identity to a rRNA sequence of a microbe selected from thegroup consisting of: Akkermansia muciniphila, Anaerostipes caccae,Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacteriuminfantis, Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridiumacetobutylicum, Clostridium aminophilum, Clostridium beijerinckii,Clostridium butyricum, Clostridium colinum, Clostridium indolis,Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii,Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobactersuccinogenes, Lactobacillus acidophilus, Lactobacillus brevis,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus,Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,Oscillospira guilliermondii, Roseburia cecicola, Roseburiainulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus,Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium,Streptococcus infantis, Streptococcus mutans, Streptococcusthermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus,Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani,Coprococcus, Coprococcus eutactus, Eubacterium cylindroides, Eubacteriumdolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis,Roseburia intestinalis, and any combination thereof.

The method of any of the preceding embodiments, wherein said metabolicdisorder is obesity.

The method of any of the preceding embodiments, wherein said metabolicdisorder is insulin insensitivity.

The method of any of the preceding embodiments, wherein said metabolicdisorder is Type 2 Diabetes Mellitus.

The method of any of the preceding embodiments, wherein said treatingresults in the subject losing weight as compared to a pre-treatmentlevel.

The method of any of the preceding embodiments, wherein said treatingresults in the subject having increased insulin sensitivity as comparedto a pre-treatment level.

The method of any of the preceding embodiments, wherein said treatingresults in the subject having reduced symptoms associated with themetabolic disorder as compared to a pre-treatment level.

The method of any of the preceding embodiments, wherein said subject isa subject enrolled in a clinical study.

The method of any of the preceding embodiments, wherein said at leastabout 85% sequence identity is selected from the group consisting of: atleast about 96%, at least about 97%, at least about 98%, at least about99%, at least about 99.5%, and at least about 99.5% sequence identity toa rRNA sequence.

The method of any of the preceding embodiments, wherein saidpharmaceutical composition is substantially free of fecal matterobtained from a subject.

The method of any of the preceding embodiments, wherein said at leastone of said microbes comprises a population of said microbes.

The method of any of the preceding embodiments, wherein said rRNAsequence is a 16S rRNA sequence.

The method of any of the preceding embodiments, wherein said rRNAsequence is a 23 S rRNA sequence.

The method of any of the preceding embodiments, wherein said rRNAsequence is both a 16S rRNA sequence and a 23S rRNA sequence.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated for oral delivery.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated for anal delivery.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated as a pill.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated as a capsule.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated in a liquid form suitable foradministration via an enema.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated as a suppository.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated in a liquid form suitable fordelivery via injection.

The method of any of the preceding embodiments, wherein thepharmaceutical composition further comprises a probiotic. In someaspects, the pharmaceutical composition may further comprise aprobiotic, said probiotic is selected from the group consisting of:Akkermansia muciniphila, Anaerostipes caccae, Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bifidobacterium infantis,Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridiumacetobutylicum, Clostridium aminophilum, Clostridium beijerinckii,Clostridium butyricum, Clostridium colinum, Clostridium indolis,Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii,Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobactersuccinogenes, Lactobacillus acidophilus, Lactobacillus brevis,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus,Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,Oscillospira guilliermondii, Roseburia cecicola, Roseburiainulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus,Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium,Streptococcus infantis, Streptococcus mutans, Streptococcusthermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus,Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani,Coprococcus, Coprococcus eutactus, Eubacterium cylindroides, Eubacteriumdolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis,Roseburia intestinalis, and any combination thereof.

The method of any of the preceding embodiments, wherein thepharmaceutical composition further comprises a prebiotic. In someaspects, the pharmaceutical composition may further comprise aprebiotic, said prebiotic is selected from the group consisting of:complex carbohydrates, complex sugars, resistant dextrins, resistantstarch, amino acids, peptides, nutritional compounds, biotin,polydextrose, fructooligosaccharide (FOS), galactooligosaccharides(GOS), inulin, starch, lignin, psyllium, chitin, chitosan, gums (e.g.guar gum), high amylose cornstarch (HAS), cellulose, β-glucans,hemi-celluloses, lactulose, mannooligosaccharides, mannanoligosaccharides (MOS), oligofructose-enriched inulin, oligofructose,oligodextrose, tagatose, trans-galactooligosaccharide, pectin, resistantstarch, xylooligosaccharides (XOS), and any combination thereof. In someaspects, said prebiotic is inulin.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is co-administered with an antibiotic.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is administered after an antibiotic. In someaspects, the method may comprise administering the pharmaceuticalcomposition after an antibiotic, wherein the pharmaceutical compositionis administered at least one hour after an antibiotic. In some aspects,the method may comprise administering the pharmaceutical compositionafter an antibiotic, wherein the pharmaceutical composition isadministered at least 2 hours after an antibiotic. In some aspects, themethod may comprise administering the pharmaceutical composition afteran antibiotic, wherein the pharmaceutical composition is administered atleast 12 hours after an antibiotic. In some aspects, the method maycomprise administering the pharmaceutical composition after anantibiotic, wherein the pharmaceutical composition is administered atleast 1 day after an antibiotic. In some aspects, the method maycomprise administering the pharmaceutical composition after anantibiotic, wherein the pharmaceutical composition is administered atleast 1 week after an antibiotic. In some aspects, the method maycomprise administering the pharmaceutical composition after anantibiotic, wherein the pharmaceutical composition is administered atleast 2 weeks after an antibiotic.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is administered after completion of anantibiotic regimen by the subject.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated as a dietary supplement.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is a biologic product.

The method of any of the preceding embodiments, further comprisingdetermining the sequence of a population of the subject's microbiome bysequencing. In some aspects, the method may further comprise determiningthe sequence of the subject's microbiome by sequencing, said sequencingcomprises sequencing the 16S rRNA. In some aspects, the method mayfurther comprise determining the sequence of the subject's microbiome bysequencing, said sequencing comprises sequencing the 23 S rRNA. In someaspects, the method may further comprise determining the sequence of thesubject's microbiome by sequencing, said sequencing comprises sequencingthe 23S and 16S rRNA. In some aspects, the method may further comprisedetermining the sequence of the subject's microbiome by sequencing, saidsequencing comprises Complete Biome Test resolution. In some aspects,said sequencing comprises long-read sequencing. In some aspects, themethod may further comprise determining the sequence of the subject'smicrobiome by sequencing, wherein the determining the sequence of thepopulation of the subject's microbiome is performed before treating thesubject with the pharmaceutical composition. In some aspects, the methodmay further comprise determining the sequence of the subject'smicrobiome by sequencing, wherein the determining the sequence of thepopulation of the subject's microbiome is performed after treating thesubject with the pharmaceutical composition.

The method of any of the preceding embodiments, further comprisingtransmitting data via machine-readable code.

The method of any of the preceding embodiments, further comprisingcomputing data via machine-readable code.

The method of any of the preceding embodiments, further comprisingstoring data via machine-readable code.

The method of any of the preceding embodiments, wherein the subject is amammal.

The method of any of the preceding embodiments, wherein the subject is alaboratory mammal.

The method of any of the preceding embodiments, wherein the subject is ahuman.

The method of any of the preceding embodiments, wherein said methodfurther comprises a companion diagnostic.

A method of producing the microbes of any of the preceding embodiments,the method comprising genetically-modifying the microbes to generaterecombinant microbes. In some aspects, the method may comprisegenetically-modifying the microbes to generate recombinant microbes,wherein an operon controls growth of the recombinant microbe.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated as an enteric-coated pill. Insome aspects, the method may comprise formulating the pharmaceuticalcomposition as an enteric-coated pill, wherein the enteric-coating isformed by a pH sensitive polymer. In some aspects, the method maycomprise formulating the pharmaceutical composition as an enteric-coatedpill, wherein the enteric-coating is formed by a pH sensitive polymer,wherein the polymer is eudragit FS30D.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated for delivery of the microbes tothe subject's ileum region.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated for delivery of the microbes tothe subject's colon region.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is formulated for delivery of the microbes tothe subject's ileum and colon region.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is delivered to the subject's ileum region.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is delivered to the subject's colon region.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is delivered to the subject's ileum and colonregion.

The method of any of the preceding embodiments, wherein thepharmaceutical composition is administered before food intake. In someaspects, the method may comprise administering the pharmaceuticalcomposition before food intake, wherein the pharmaceutical compositionis administered at least one hour before food intake. In some aspects,the method may comprise administering the pharmaceutical compositionbefore food intake, wherein the pharmaceutical composition isadministered at least 2 hours before food intake. In some aspects, themethod may comprise administering the pharmaceutical composition beforefood intake, wherein the pharmaceutical composition is administered atleast 3 hours before food intake. In some aspects, the method maycomprise administering the pharmaceutical composition before foodintake, wherein the pharmaceutical composition is administered at least4 hours before food intake.

The method of any of the preceding embodiments, wherein said microbesare administered with food intake.

The method of any of the preceding embodiments, wherein said microbescomprise a synergistic stability in the pharmaceutical composition ascompared to individual strains.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

The content of the International Nucleotide Sequence DatabaseCollaboration (DDBJ/EMBL/GENBANK) accession number CP001071.1 formicrobial strain Akkermansia muciniphila, culture collection ATCCBAA-835, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AJ518871.2 formicrobial strain Anaerofustis stercorihominis, culture collection DSM17244, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number DS499744.1 formicrobial strain Anaerostipes caccae, culture collection DSM 14662, isherein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AJ270487.2 formicrobial strain Anaerostipes caccae, butyrate-producing bacteriumL1-92, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AY305319.1 formicrobial strain Anaerostipes hadrus, butyrate-producing bacteriumSS2/1, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AJ315980.1 formicrobial strain Anaerotruncus colihominis, culture collection DSM17241, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AP009256.1 formicrobial strain, Bifidobacterium adolescentis, culture collection ATCC15703, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number CP001095.1 formicrobial strain Bifidobacterium longum subsp. infantis, culturecollection ATCC 15697, is herein incorporated by reference in itsentirety.

The content of DDBFEMBL/GenBank accession number U41172.1 for microbialstrain Butyrivibrio fibrisolvens, culture collection ATCC 19171, isherein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AJ250365.2 formicrobial strain Butyrivibrio fibrisolvens, 16.4, is herein incorporatedby reference in its entirety.

The content of DDBFEMBL/GenBank accession number U41168.1 for microbialstrain Butyrivibrio fibrisolvens, OB156, is herein incorporated byreference in its entirety.

The content of DDBFEMBL/GenBank accession number AY305305.1 formicrobial strain Butyrate-producing bacterium, A2-232, is hereinincorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AY305316.1 formicrobial strain Butyrate-producing bacterium, SS3/4, is hereinincorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AE001437.1 formicrobial strain Clostridium acetobutylicum, culture collection ATCC824, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number X78070.1 for microbialstrain Clostridium acetobutylicum, culture collection DSM 792, is hereinincorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number CP000721.1 formicrobial strain Clostridium beijerinckii, culture collection NCIMB8052, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number X68189.1 for microbialstrain Clostridium sporogenes, is herein incorporated by reference inits entirety.

The content of DDBJ/EMBL/GENBANK accession number X74770.1 for microbialstrain Clostridium tetani, is herein incorporated by reference in itsentirety.

The content of DDBJ/EMBL/GENBANK accession number AJ270491.2 formicrobial strain Coprococcus, butyrate-producing bacterium L2-50, isherein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number EF031543.1 formicrobial strain Coprococcus eutactus, culture collection ATCC 27759, isherein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AY305306.1 formicrobial strain Eubacterium cylindroides, butyrate-producing bacteriumT2-87, is herein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AY305313.1 formicrobial strain Eubacterium cylindroides, butyrate-producing bacteriumSM7/11, is herein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number L34682.2 for microbialstrain Eubacterium dolichum, culture collection DSM 3991, is hereinincorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AJ270490.2 formicrobial strain Eubacterium halii, butyrate-producing bacterium L2-7,is herein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AY305318.1 formicrobial strain Eubacterium halii, butyrate-producing bacterium SM6/1,is herein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number L34621.2 for microbialstrain Eubacterium halii, culture collection ATCC 27751, is hereinincorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AJ270475.2 formicrobial strain Eubacterium rectale, A1-86, is herein incorporated byreference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number NC 012781.1 formicrobial strain Eubacterium rectale, culture collection ATCC 33656, isherein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number L34421.2 for microbialstrain Eubacterium ventriosum, culture collection ATCC 27560, is hereinincorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AY305307.1 formicrobial strain Faecalibacterium prausnitzii, butyrate producingbacterium M21/2, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number FP929046.1 formicrobial strain Faecalibacterium prausnitzii is herein incorporated byreference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number GG697168.2 formicrobial strain Faecalibacterium prausnitzii is herein incorporated byreference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number CP002158.1 formicrobial strain Fibrobacter succinogenes subsp. succinogenes is hereinincorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number NZ AUJNO1000001.1 formicrobial strain Clostridium butyricum is herein incorporated byreference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number NZ AZUI01000001.1 formicrobial strain Clostridium indolis, culture collection DSM 755, isherein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number ACEP01000175.1 formicrobial strain Eubacterium hallii, culture collection DSM 3353, isherein incorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AY305310.1 formicrobial strain Roseburia faecis, M72/1, is herein incorporated byreference in its entirety.

The content of DDBFEMBL/GenBank accession number AJ270482.2 formicrobial strain Roseburia hominis, type strain A2-183T, is hereinincorporated by reference in its entirety.

The content of DDBFEMBL/GenBank accession number AJ312385.1 formicrobial strain Roseburia intestinalis, L1-82, is herein incorporatedby reference in its entirety.

The content of DDBFEMBL/GenBank accession number AJ270473.3 formicrobial strain Roseburia inulinivorans, type strain A2-194T, is hereinincorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number NZ ACFY01000179.1 formicrobial strain Roseburia inulinivorans, culture collection DSM 16841,is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number K1912489.1 formicrobial strain Ruminococcus flavefaciens, culture collection ATCC19208, is herein incorporated by reference in its entirety.

The content of DDBJ/EMBL/GENBANK accession number AAYG02000043.1 formicrobial strain Ruminococcus gnavus, culture collection ATCC 29149, isherein incorporated by reference in its entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts exemplary microbiome-related health conditions anddiseases for which microbiome therapeutics and diagnostics can be used.These health conditions can include: preterm labor, chronic fatiguesyndrome, skin health (e.g. acne), Type 2 Diabetes Mellitus (T2DM),allergies, depression, autism, asthma, hypertension, irritable bowelsyndrome, metabolism, obesity, drug metabolism, vaginosis, atopicdermatitis, psoriasis, Type I Diabetes (T1DM), Multiple Sclerosis,Clostridium Difficile, Inflammatory Bowel Disease (IBD), Crohn'sDisease, genitourinary disorders, and heart disease.

FIG. 2 depicts an illustrative microbiome mediated pathway that can beaffected to create a Type 2 Diabetes Mellitus (T2DM) therapeutic. Aformulation comprising a prebiotic (e.g. inulin), a primary fermenter(e.g. Bifidobacterium), and a secondary fermenter (e.g. Clostridiumand/or Eubacterium) can be used for butyrate production.

FIG. 3 is an illustration depicting an exemplary platform for a CompleteBiome Test (CBT) (e.g. as a diagnostic test or as a development tool todevelop therapeutics). The specific microbiotic actionable targetsstarting with microbiotic strains obtained from, e.g. fecal mattertransplants (FMT), the microorganism(s), the genus, and thepresence/absence of microorganism strain(s) related to health conditionsor diseases can be determined using the Complete Biome Test.

FIG. 4(A) depicts the microbiome strain resolution using standard testsand (B) the increased microbiome strain resolution using the CompleteBiome Test.

FIG. 5 depicts an illustrative process for generating a database usingdata obtained from the group consisting of: external data (e.g.scientific literature and/or databases), patient information, measuredepigenetic changes, measured functional pathways, measured strainclassification, and any combinations thereof. The database can be used,e.g. to drive identification of a therapeutic consortia (e.g. fortreatment of health conditions or diseases).

FIG. 6 depicts an exemplary process used to identify strains related toT2DM (e.g. to identify a therapeutic consortia). Exemplary T2DM strainsfound using this method include: Butyrivibrio fibrisolvens,Streptococcus mutans, Ruminococcus gnavus, Roseburia cecicola,Fibrobacter succinogenes, Ruminococcus flavefaciens, and Clostridiumindolis.

FIG. 7 depicts a system adapted to enable a user to detect, analyze, andprocess data (e.g. sequencing data, strain classification, functionalpathways, epigenetic changes, patient information, external data,databases, microbiome strains; therapeutic consortia, etc.) usingmachine readable code.

FIG. 8 depicts how both the diagnostic and therapeutic approach outlinedherein can comprise a targeted microbe strain selection as compared to acomposite fecal microbiome transplant.

FIG. 9 depicts how by combining strains together in a formulation thestability of all of the individual strains either can remain high ordramatically improve. Stability of individual strains, Clostridiumbutyricum (CBUT), Clostridium beijerinckii (CBEI), Bifidobacteriumlongum (BLON), and Bifidobacterium infantis (BINF) was compared withformulations WB00002 and WB00003, which can comprise strains CBUT, CBEI,BLON, BINF present together in the formulation. The formulations canadditionally comprise strains B. adolescentis, A. muciniphila, E.hallii, and C. indolis. Formulations WB0002 and WB0003 differ in thatWB0003 can also comprise a prebiotic in combination with the strains.The increased stability of the formulations suggests that theformulation can provide a synergistic stability when the strains aretogether over individual strains.

FIG. 10 depicts how formulation WB0003 can result in increased weightloss during the dosing period of a preclinical study involvingdiet-induced obese mice. Support of this effect is further corroboratedby the weight regain during the washout period. Linagliptin is apositive control.

FIG. 11(A) depicts a bi-modal response in glucose control as measured byArea Under the Oral Glucose Tolerance Test (OGTT) Curve (AUC) for micedosed with formulation WB0003. (B) Depicts the OGTT curves that comparethe responders to WB0003, to control, and to the positive control,Linagliptin.

FIG. 12 illustrates that de novo assembly for C. butyricum using methodsof the invention can result in the use of less contigs (e.g., 2 contigs)than those found in the database (e.g., 40 contigs).

FIG. 13(A) illustrates that de novo assembly using a method of theinvention can differentiate between several operon orderings and forthis example strain of C. butyricum a ‘type C’ ordering was discovered.(B) Tabulates the exact genomic coordinates for five of the butyratepathway genes for this strain.

FIG. 14 illustrates exemplary data for short chain fatty acidquantification in different media (e.g., RCM, PYG) by strain. The shortchain fatty acid quantification shows that the predicted genomicfunction of the strains matches the actual function. This can be similarfor different media. In one non-limiting example, strain 1 can beBifidobacterium adolescentis (BADO). In one non-limiting example, strain2 is Bifidobacterium infantis (BINF). In one non-limiting example,strain 3 is Bifidobacterium longum (BLON). In one non-limiting example,strain 4 is Clostridium beijerinckii (CBEI). In one non-limitingexample, strain 5 is Clostridium butyricum (CBUT). In one non-limitingexample, strain 6 is Clostridium indolis (CIND). In one non-limitingexample, strain 7 is Eubacterium hallii (EHAL).

FIG. 15 illustrates that improved media of the invention (e.g.,PYGveg+vit+salt+buffer) can result in higher peak bacterial density. Inone non-limiting example, strain 1 is Akkermansia muciniphila (AMUC). Inone non-limiting example, strain 2 is CBEI. In one non-limiting example,strain 3 is EHAL. In one non-limiting example, strain 4 is CIND. In onenon-limiting example, strain 5 is BLON. In one non-limiting example,strain 6 is BADO. In one non-limiting example, strain 7 is CBUT. In onenon-limiting example, strain 8 is BINF.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a sample” includes a plurality ofsamples, including mixtures thereof.

The terms “microbes” and “microorganisms” are used interchangeablyherein and can refer to bacteria, archaea, eukaryotes (e.g. protozoa,fungi, yeast), and viruses, including bacterial viruses (i.e. phage).

The term “microbiome”, “microbiota”, and “microbial habitat” are usedinterchangeably herein and can refer to the ecological community ofmicroorganisms that live on or in a subject's body. The microbiome canbe comprised of commensal, symbiotic, and/or pathogenic microorganisms.Microbiomes can exist on or in many, if not most parts of the subject.Some non-limiting examples of habitats of microbiome can include: bodysurfaces, body cavities, body fluids, the gut, the colon, skin surfacesand pores, vaginal cavity, umbilical regions, conjunctival regions,intestinal regions, the stomach, the nasal cavities and passages, thegastrointestinal tract, the urogenital tracts, saliva, mucus, and feces.

The term “prebiotic” as used herein can be a general term to refer tochemicals and or ingredients that can affect the growth and/or activityof microorganisms in a host (e.g. can allow for specific changes in thecomposition and/or activity in the microbiome). Prebiotics can confer ahealth benefit on the host. Prebiotics can be selectively fermented,e.g. in the colon. Some non-limiting examples of prebiotics can include:complex carbohydrates, complex sugars, resistant dextrins, resistantstarch, amino acids, peptides, nutritional compounds, biotin,polydextrose, fructooligosaccharide (FOS), galactooligosaccharides(GOS), inulin, lignin, psyllium, chitin, chitosan, gums (e.g. guar gum),high amylose cornstarch (HAS), cellulose, β-glucans, hemi-celluloses,lactulose, mannooligosaccharides, mannan oligosaccharides (MOS),oligofructose-enriched inulin, oligofructose, oligodextrose, tagatose,trans-galactooligosaccharide, pectin, resistant starch, andxylooligosaccharides (XOS). Prebiotics can be found in foods (e.g.acacia gum, guar seeds, brown rice, rice bran, barley hulls, chicoryroot, Jerusalem artichoke, dandelion greens, garlic, leek, onion,asparagus, wheat bran, oat bran, baked beans, whole wheat flour,banana), and breast milk. Prebiotics can also be administered in otherforms (e.g. capsule or dietary supplement).

The term “probiotic” as used herein can mean one or more microorganismswhich, when administered appropriately, can confer a health benefit onthe host or subject. Some non-limiting examples of probiotics include:Akkermansia muciniphila, Anaerostipes caccae, Bifidobacteriumadolescentis, Bifidobacterium bifidum, Bifidobacterium infantis,Bifidobacterium longum, Butyrivibrio fibrisolvens, Clostridiumacetobutylicum, Clostridium aminophilum, Clostridium beijerinckii,Clostridium butyricum, Clostridium colinum, Clostridium indolis,Clostridium orbiscindens, Enterococcus faecium, Eubacterium hallii,Eubacterium rectale, Faecalibacterium prausnitzii, Fibrobactersuccinogenes, Lactobacillus acidophilus, Lactobacillus brevis,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus caucasicus,Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis,Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus,Oscillospira guilliermondii, Roseburia cecicola, Roseburiainulinivorans, Ruminococcus flavefaciens, Ruminococcus gnavus,Ruminococcus obeum, Streptococcus cremoris, Streptococcus faecium,Streptococcus infantis, Streptococcus mutans, Streptococcusthermophilus, Anaerofustis stercorihominis, Anaerostipes hadrus,Anaerotruncus colihominis, Clostridium sporogenes, Clostridium tetani,Coprococcus, Coprococcus eutactus, Eubacterium cylindroides, Eubacteriumdolichum, Eubacterium ventriosum, Roseburia faeccis, Roseburia hominis,Roseburia intestinalis, and any combination thereof.

The terms “determining”, “measuring”, “evaluating”, “assessing,”“assaying,” and “analyzing” can be used interchangeably herein and canto refer to any form of measurement, and include determining if anelement is present or not. (e.g., detection). These terms can includeboth quantitative and/or qualitative determinations. Assessing may berelative or absolute. These terms can include use of the algorithms anddatabases described herein. “Detecting the presence of” can includedetermining the amount of something present, as well as determiningwhether it is present or absent. The term “genome assembly algorithm” asused herein, refers to any method capable of aligning sequencing readswith each other (de novo) or to a reference (re-sequencing) underconditions that a complete sequence of the genome may be determined.

The term “genome” as used herein, can refer to the entirety of anorganism's hereditary information that is encoded in its primary DNAsequence. The genome includes both the genes and the non-codingsequences. For example, the genome may represent a microbial genome. Thegenetic content of the microbiome can comprise: genomic DNA, RNA, andribosomal RNA, the epigenome, plasmids, and all other types of geneticinformation found in the microbes that comprise the microbiome.

“Nucleic acid sequence” and “nucleotide sequence” as used herein referto an oligonucleotide or polynucleotide, and fragments or portionsthereof, and to DNA or RNA of genomic or synthetic origin which may besingle- or double-stranded, and represent the sense or antisense strand.The nucleic acid sequence can be made up of adenine, guanine, cytosine,thymine, and uracil (A, T, C, G, and U) as well as modified versions(e.g. N6-methyladenosine, 5-methylcytosine, etc.).

The terms “homology” and “homologous” as used herein in reference tonucleotide sequences refer to a degree of complementarity with othernucleotide sequences. There may be partial homology or complete homology(i.e., identity). A nucleotide sequence which is partiallycomplementary, i.e., “substantially homologous,” to a nucleic acidsequence is one that at least partially inhibits a completelycomplementary sequence from hybridizing to a target nucleic acidsequence.

The term “sequencing” as used herein refers to sequencing methods fordetermining the order of the nucleotide bases—A, T, C, G, and U—in anucleic acid molecule (e.g., a DNA or RNA nucleic acid molecule.

The term “biochip” or “array” can refer to a solid substrate having agenerally planar surface to which an adsorbent is attached. A surface ofthe biochip can comprise a plurality of addressable locations, each ofwhich location may have the adsorbent bound there. Biochips can beadapted to engage a probe interface, and therefore, function as probes.Protein biochips are adapted for the capture of polypeptides and can becomprise surfaces having chromatographic or biospecific adsorbentsattached thereto at addressable locations. Microarray chips aregenerally used for DNA and RNA gene expression detection.

The term “barcode” as used herein, refers to any unique, non-naturallyoccurring, nucleic acid sequence that may be used to identify theoriginating genome of a nucleic acid fragment.

The terms “subject,” “individual,” “host,” and “patient” can be usedinterchangeably herein and refer to any animal subject, including:humans, laboratory animals, livestock, and household pets. The subjectcan host a variety of microorganisms. The subject can have differentmicrobiomes in various habitats on and in their body. The subject may bediagnosed or suspected of being at high risk for a disease. The subjectmay have a microbiome state that is contributing to a disease (adysbiosis). In some cases, the subject is not necessarily diagnosed orsuspected of being at high risk for the disease. In some instances asubject may be suffering from an infection or at risk of developing ortransmitting to others an infection.

The terms “treatment” or “treating” are used interchangeably herein.These terms can refer to an approach for obtaining beneficial or desiredresults including but not limited to a therapeutic benefit and/or aprophylactic benefit. A therapeutic benefit can mean eradication oramelioration of the underlying disorder being treated. Also, atherapeutic benefit can be achieved with the eradication or ameliorationof one or more of the physiological symptoms associated with theunderlying disorder such that an improvement is observed in the subject,notwithstanding that the subject may still be afflicted with theunderlying disorder. A prophylactic effect includes delaying,preventing, or eliminating the appearance of a disease or condition,delaying or eliminating the onset of symptoms of a disease or condition,slowing, halting, or reversing the progression of a disease orcondition, or any combination thereof. For prophylactic benefit, asubject at risk of developing a particular disease, or to a subjectreporting one or more of the physiological symptoms of a disease mayundergo treatment, even though a diagnosis of this disease may not havebeen made.

The terms “16S”, “16S ribosomal subunit”, and “16S ribosomal RNA (rRNA)”can be used interchangeably herein and can refer to a component of asmall subunit (e.g., 30S) of a prokaryotic (e.g., bacteria, archaea)ribosome. The 16S rRNA is highly conserved evolutionarily among speciesof microorganisms. Consequently, sequencing of the 16S ribosomal subunitcan be used to identify and/or compare microorganisms present in asample (e.g., a microbiome).

The terms “23S”, “23 S ribosomal subunit”, and “23 S ribosomal RNA(rRNA)” can be used interchangeably herein and can refer to a componentof a large subunit (e.g., 50S) of a prokaryotic (e.g., bacteria,archaea) ribosome. Sequencing of the 23S ribosomal subunit can be usedto identify and/or compare microorganisms present in a sample (e.g., amicrobiome).

The term “spore” as used herein can refer to a viable cell produced by amicroorganism to resist unfavorable conditions such as hightemperatures, humidity, and chemical agents. A spore can have thickwalls that allow the microorganism to survive harsh conditions forextended periods of time. Under suitable environmental conditions, aspore can germinate to produce a living form of the microorganism thatis capable of reproduction and all of the physiological activities ofthe microorganism.

Overview

Compositions comprising microbes such as probiotics can confer a varietyof beneficial effects on a subject. Examples of these beneficial effectscan include immunomodulatory features, regulation of cell proliferation,the ability to promote normal physiologic development of the mucosalepithelium, and enhancement of human nutrition. Microbial-basedcompositions can be administered as a therapeutic to a subject sufferingfrom a microbiome-related health condition or disorder.

In some embodiments, the disclosure provides a diagnostic assay forpredicting a disease status of a subject or likelihood of a subject'sresponse to a therapeutic. The diagnostic assay can use the presence ofone or more microbes in a sample or a microbiome profile of a subject tocalculate a quantitative score. The quantitative score can be used topredict disease status or likelihood of response to a therapeutic in asubject. In some applications, the diagnostic assay can use the presenceof one or more microbes and one or more characteristics, such as, e.g.,age, weight, gender, medical history, risk factors, family history, or acombination thereof to calculate a quantitative score that can be usedto predict disease status or likelihood of response to a therapeutic ina subject. In some applications, the diagnostic assay can further useenvironmental factors such as geographic location, type of work, and useof hygiene products to calculate a quantitative score.

An exemplary method of the disclosure can comprise at least one of thefollowing steps: obtaining a biological sample from a subject, measuringa panel of microbes in the biological sample of the subject, determininga disease status upon the measuring, generating a report that providesinformation of disease status upon the results of the determining, andadministering microbial-based compositions of the disclosure to thesubject for preventing and/or treating a health condition such as amicrobiome-based disorder, or the presence or absence of a microbe.

Methods for Determining Members of a Microbial Habitat

The present disclosure provides methods and compositions comprisingmicrobial populations for the treatment of microbiome-related healthconditions and/or disorders in a subject. Methods of the disclosure caninclude collection, stabilization and extraction of microbes formicrobiome analysis. Methods of the disclosure can include determiningthe composition of a microbial habitat of a host to generate amicrobiome profile. The composition of a microbial habitat can be usedto diagnose a health condition of a host, for example, to determinelikelihood of a disorder and/or treatment course of the disorder.

In some embodiments, methods of the disclosure can be used to determinemicrobial habitat of the gut or gastrointestinal tract of a subject. Thegut comprises a complex microbiome including multiple species ofmicrobes that can contribute to vitamin production and absorption,metabolism of proteins and bile acids, fermentation of dietarycarbohydrates, and prevention of pathogen overgrowth. The composition ofmicrobes within the gut can be linked to functional metabolic pathwaysin a subject. Non-limiting examples of metabolic pathways linked to gutmicrobiota include, energy balance regulation, secretion of leptin,lipid synthesis, hepatic insulin sensitivity, modulation of intestinalenvironment, and appetite signaling. Modification of the gut microbiomecan increase the risk for health conditions such as ulcerative colitis,colorectal cancer, autoimmune disorders, obesity, diabetes, andinflammatory bowel disease.

In some embodiments, detection methods (e.g. sequencing) can be used toidentify gut microbiome biomarkers associated with, for example, obesityand obesity-induced diabetes. For example, non-obese and obese subjectscan be categorized based on differences in species of microbes presentin their microbiome. Obese subjects can have reduced microbial diversityand higher levels of fermentation causing microbes, for example,bacteroidetes phylum and methanogenic archaea, compared with non-obesesubjects. Subjects with obesity-induced diabetes can have a microbiotathat promotes mass gain, metabolic endotoxemia, adipose tissueinflammation, and insulin resistance. Differences in microbes betweenobese and lean subjects can be used to generate microbial biomarkerprofiles associated with obesity that can be used to predict riskfactors and/or treatment course.

In some embodiments, detection methods of the disclosure (e.g.,sequencing) can be used to analyze changes in gut microbiome compositionover time, for example, during antibiotic treatment, gut microbiometherapies, and various diets. The microbiome can be significantlyaltered upon exposure to antibiotics and diets that deplete the nativemicrobial population. Methods of the disclosure can be used to generateprofiles of the subject before and after administration of a therapeuticto characterize differences in the microbiota.

In some embodiments, methods to visualize the microbiome based onsequencing signatures are provided. In some embodiments, methods areprovided to visualize the microbiome over time based on sequencinginformation.

Methods of the disclosure can be used to detect, characterize andquantify microbial habitat of the amniotic fluid of a pregnant woman.The amniotic cavity of a pregnant woman undergoing preterm labor canharbor genetic material from a greater diversity of microbes, includingpreviously-uncharacterized microbes, compared with pregnant womandelivering at full-term. The microbial habit can be used to define thediversity and abundance of microbes invading the amniotic cavity inorder to evaluate clinical significance and causal framework for pretermlabor. The microbiome profiles of amniotic fluid of women with full-termdelivery and preterm delivery can be compared to determine microbes thatcan be used as biomarkers for predicting and/or treating preterm labor.

Microorganisms can translocate from a mother to an infant throughmaternal mononuclear cells in breast milk, which may prime thedeveloping infant immune system to appropriately respond to commensaland pathogenic bacteria. Methods of the disclosure can be used todetermine microbial habitat of the gut of an infant to generate patternsof microbial colonization and effects of the microbes on development ofimmunity during infancy and early childhood.

Methods of the disclosure can be used to analyze microbial habitat ofthe skin. Parts of the skin, including cutaneous invaginations andappendages, sweat glands (eccrine and apocrine), sebaceous glands andhair follicles, can each be associated with unique microbiota.Comparison of skin microbiome profiles of a healthy subject and asubject with for example, acne, can provide insights into microbialinvolvement in skin health and disease.

Biological Samples

A biological sample can be collected from a subject to determine themicrobiome profile of the subject. The biological sample can be anysample type from any microbial habitat on the body of a subject.Non-limiting examples of microbial habitats include skin habitat,umbilical habitat, vaginal habitat, amniotic fluid habitat, conjunctivalhabitat, intestinal habitat, stomach habitat, gut habitat, oral habitat,nasal habitat, gastrointestinal tract habitat, respiratory habitat, andurogenital tract habitat.

Depending on the application, the selection of a biological sample canbe tailored to the specific application. The biological sample can befor example, whole blood, serum, plasma, mucosa, saliva, cheek swab,urine, stool, cells, tissue, bodily fluid, lymph fluid, CNS fluid, andlesion exudates. A combination of biological samples can be used withthe methods of the disclosure.

Sample Preparation

Sample preparation can comprise any one of the following steps or acombination of steps. A sterile swab is first dipped into a tubecontaining sterile phosphate buffered saline (PBS) to wet. The swab isswiped across the area of interest multiple times (e.g., 10-20 times)with enough vigor that the tissue is slightly pink/red coloredafterwards. The swab is gently dipped into a buffer (e.g., a lysisbuffer) in a sterile tube. The swab is left in the tube for shipping toa laboratory to be further analyzed as provided herein. The samplesobtained can be shipped overnight at room temperature.

Shipping microbial cells in buffers can introduce detection bias in thesamples. Some microbes can continue propagating on the nutrients thatcome along with sample collection. Some microbes can undergo apoptosisin the absence of a specific environment. As a result, microbial samplesshipped in this fashion can have an initial profiling/population biasassociated with cellular integrity.

Methods can be used to enrich intact cells by first centrifuging thecollected sample. The resulting pellet, formed from the intact cellswithin the sample, can then be used as a precursor for all of thedownstream steps. In some embodiments, the methods of the disclosurefurther comprise a purification step to concentrate any DNA present inthe supernatant (e.g. from already lysed cells). This DNA can becombined with DNA extracted from the standard pellet preparation. Thecombined DNA can form a more complete precursor to the downstream steps.

Cell lysis and/or extraction of nucleic acids from the cells can beperformed by any suitable methods including physical methods, chemicalmethods, or a combination of both. Nucleic acids can be isolated from abiological sample using shearing methods, which preserve the integrityand continuity of genomic DNA.

A nucleic acid sample used with the present disclosure can include alltypes of DNA and RNA. The length of nucleic acids can be about 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000,600,000, 700,000, 800,000, 900,000, 1,000,000, 2,000,000, 3,000,000,4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, or10,000,000, nucleotides or base pairs in length.

An amplicon approach can be used to prepare DNA for microbiomeprofiling. This approach can comprise a number of steps, for example,PCR, sample quantification (e.g. Qubit, nanodrop, bioanalyzer, etc.),Blue Pippin size selection, 0.5× Ampure purification, samplequantification, DNA end repair, 0.5× Ampure purification, blunt endadaptor ligation, exo-nuclease treatment, two 0.5× Ampure purifications,and final Blue Pippen size selection.

In some embodiments, the method does not use an amplification step.Examples of such methods include preparation of samples for sequencingby Whole Genome Shotgun (WGS) sequencing. These approaches can provide abenefit by removing amplification bias that can skew microbialdistributions. In addition, such approaches can allow for de novodiscovery of pertinent elements, for example, bacterial plasmids, fungiand viruses.

The practice of the methods of the present disclosure can employconventional techniques of immunology, biochemistry, chemistry,molecular biology, microbiology, cell biology, genomics and recombinantDNA, which are within the skill of the art. For example, preparation ofa biological sample can comprise, e.g., extraction or isolation ofintracellular material from a cell or tissue such as the extraction ofnucleic acids, protein, or other macromolecules. Sample preparationwhich can be used with the methods of disclosure include but are notlimited to, centrifugation, affinity chromatography, magneticseparation, immunoassay, nucleic acid assay, receptor-based assay,cytometric assay, colorimetric assay, enzymatic assay, electrophoreticassay, electrochemical assay, spectroscopic assay, chromatographicassay, microscopic assay, topographic assay, calorimetric assay,radioisotope assay, protein synthesis assay, histological assay, cultureassay, and combinations thereof.

Microbiome Profiling

The present disclosure provides methods for measuring at least onemicrobe in a biological sample from at least one microbial habitat of asubject and determining a microbiome profile. A microbiome profile canbe assessed using any suitable detection means that can measure orquantify one or more microbes (bacteria, fungi, viruses and archaea)that comprise a microbiome.

A Complete Biome Test (CBT) can generate microbiome profiles with, forexample, strain-level resolution. A CBT can be performed usingmicrobiome profiling methods described herein. FIG. 3 provides anillustration depicting an exemplary platform for a CBT (e.g. as adiagnostic test or as a development tool to develop therapeutics). Thespecific microbiotic actionable targets starting with microbioticstrains obtained from, e.g. fecal matter transplants (FMT), themicroorganism(s), the genus, and the presence/absence of microorganismstrain(s) related to health conditions or diseases can be determinedusing the CBT. FIG. 4(A) depicts the microbiome strain resolution usingstandard tests. FIG. 4(B) depicts the increased microbiome strainresolution using the CBT. FIG. 5 depicts an illustrative process forgenerating a database (e.g., a CBT driven-database using data obtainedfrom the group consisting of: external data (e.g. scientific literatureand/or databases), patient information, measured epigenetic changes,measured functional pathways, measured strain classification, and anycombinations thereof. The database can be used, e.g. to driveidentification of a therapeutic consortia (e.g. for treatment of healthconditions or diseases). FIG. 8 depicts how both the diagnostic andtherapeutic approach outlined herein can comprise a targeted microbestrain selection or therapeutic consortia as compared to a compositefecal microbiome transplant.

Nucleic acid sample prepared from a biological sample can be subjectedto a detection method to generate a profile of the microbiome associatedwith the sample. Profiling of a microbiome can comprise one or moredetection methods.

Methods of the disclosure can be used to measure, for example, a 16Sribosomal subunit, a 23 S ribosomal subunit, intergenic regions, andother genetic elements. Suitable detection methods can be chosen toprovide sufficient discriminative power in a particular microbe in orderto identify informative microbiome profiles.

In some applications, the entire genomic region of the 16S or 23Sribosomal subunit of the microbe is analyzed to determine a subject'smicrobiome profile. In some applications, the variable regions of the16S and/or 23S ribosomal subunit of the microbe are analyzed todetermine a subject's microbiome profile.

In some applications, the entire genome of the microbe is analyzed todetermine a subject's microbiome profile. In other applications, thevariable regions of the microbe's genome are analyzed to determine asubject's microbiome profile. For example, genetic variation in thegenome can include restriction fragment length polymorphisms, singlenucleotide polymorphisms, insertions, deletions, indels(insertions-deletions), microsatellite repeats, minisatellite repeats,short tandem repeats, transposable elements, randomly amplifiedpolymorphic DNA, amplification fragment length polymorphism or acombination thereof.

In some embodiments, sequencing methods such as long-read length singlemolecule sequencing is used for detection. Long read sequencing canprovide microbial classification down to the strain resolution of eachmicrobe. Examples of sequencing technologies that can be used with thepresent disclosure for achieving long read lengths include the SMRTsequencing systems from Pacific Biosciences, long read length Sangersequencing, long read ensemble sequencing approaches, e.g.,Illumina/Moleculo sequencing and potentially, other single moleculesequencing approaches, such as Nanopore sequencing technologies.

Long read sequencing can include sequencing that provides a contiguoussequence read of for example, longer than 500 bases, longer than 800bases, longer than 1000 bases, longer than 1500 bases, longer than 2000bases, longer than 3000 bases, or longer than 4500 bases.

In some embodiments, detection methods of the disclosure compriseampification-mode sequencing to profile the microbiome. In someembodiments, detection methods of the disclosure comprise anon-amplification mode, for example Whole Genome Shotgun (WGS)sequencing, to profile the microbiome.

Primers used in the disclosure can be prepared by any suitable method,for example, cloning of appropriate sequences and direct chemicalsynthesis. Primers can also be obtained from commercial sources. Inaddition, computer programs can be used to design primers. Primers cancontain unique barcode identifiers.

Microbiome profiling can further comprise use of for example, a nucleicacid microarray, a biochip, a protein microarray, an analytical proteinmicroarray, reverse phase protein microarray (RPA), a digital PCRdevice, and/or a droplet digital PCR device.

In some embodiments, the microbial profile is determined usingadditional information such as age, weight, gender, medical history,risk factors, family history, or any other clinically relevantinformation.

In some applications, a subject's microbiome profile comprises a singlemicrobiome. For example, a subject's microbiome profile can comprise ofat least one biological sample from only the subject's intestinalmicrobiome. In another example, a subject's microbiome profile cancomprise of at least one biological sample from only the subject'sstomach microbiome. In another example, a subject's microbiome profilecan comprise of at least one biological sample from only the subject'sgut microbiome. In another example, a subject's microbiome profile cancomprise of at least one biological sample from only the subject's oralmicrobiome.

In some applications, a subject's microbiome profile comprises at leastone biological sample from more than one microbiome. For example, asubject's microbiome profile can comprise of at least one biologicalsample from the subject's skin microbiome and at least one biologicalsample from the umbilical microbiome. In another example, a subject'smicrobiome profile can comprise of at least one biological sample fromthe subject's intestinal microbiome, at least one biological sample fromthe stomach microbiome, at least one biological sample from the gutmicrobiome, and at least one biological sample from the oral microbiome.In another example, a subject's microbiome profile can comprise of atleast one biological sample from the subject's intestinal microbiome,and at least one biological sample from stomach microbiome. In anotherexample, a subject's microbiome profile can comprise of at least onebiological sample from the subject's gut microbiome, and at least onebiological sample from oral microbiome. In some applications, asubject's microbiome profile can comprise of at least 2, 3, 4, 5, 6, 7,8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18,19, 20 microbiomes.

A subject's microbiome profile can comprise of one microbe. In someapplications, a subject's microbiome profile comprises of, for example,2 microbes, 3 or fewer microbes, 4 or fewer microbes, 5 or fewermicrobes, 6 or fewer microbes, 7 or fewer microbes, 8 or fewer microbes,9 or fewer microbes, 10 or fewer microbes, 11 or fewer microbes, no morethan 12 microbes, 13 or fewer microbes, 14 or fewer microbes, 15 orfewer microbes, 16 or fewer microbes, 18 or fewer microbes, 19 or fewermicrobes, 20 or fewer microbes, 25 or fewer microbes, 30 or fewermicrobes, 35 or fewer microbes, 40 or fewer microbes, 45 or fewermicrobes, 50 or fewer microbes, 55 or fewer microbes, 60 or fewermicrobes, 65 or fewer microbes, 70 or fewer microbes, 75 or fewermicrobes, 80 or fewer microbes, 85 or fewer microbes, 90 or fewermicrobes, 100 or fewer microbes, 200 or fewer microbes, 300 or fewermicrobes, 400 or fewer microbe, 500 or fewer microbes, 600 or fewermicrobes, 700 or fewer microbes, or 800 or fewer microbes.

Algorithm-Based Methods

The present disclosure provides algorithm-based methods for building amicrobiome profile of a subject. Non-limiting examples of algorithmsthat can be used with the disclosure include elastic networks, randomforests, support vector machines, and logistic regression.

The algorithms can transform the underlying measurements into aquantitative score or probability relating to, for example, diseaserisk, disease likelihood, presence or absence of disease, presence orabsence of a microbe, treatment response, and/or classification ofdisease status. The algorithms can aid in the selection of importantmicrobes.

Analysis

A microbiome profile of a subject can be analyzed to determineinformation related to the health status of the subject. The informationcan include, for example, degree of likelihood of a disorder, presenceor absence of a disease state, a poor clinical outcome, good clinicaloutcome, high risk of disease, low risk of disease, complete response,partial response, stable disease, non-response, and recommendedtreatments for disease management.

The analysis can be a part of a diagnostic assay to predict diseasestatus of a subject or likelihood of a subject's response to atherapeutic. The diagnostic assay can use the quantitative scorecalculated by the algorithms-based methods described herein to performthe analysis.

In some applications, an increase in one or more microbes' thresholdvalues or quantitative score in a subject's microbiome profile indicatesan increased likelihood of one or more of: a poor clinical outcome, goodclinical outcome, high risk of disease, low risk of disease, completeresponse, partial response, stable disease, non-response, andrecommended treatments for disease management. In some embodiments, adecrease in the quantitative score indicates an increased likelihood ofone or more of: a poor clinical outcome, good clinical outcome, highrisk of disease, low risk of disease, complete response, partialresponse, stable disease, non-response, and recommended treatments fordisease management.

In some applications, a decrease in one or more microbes' thresholdvalues or quantitative score in a subject's microbiome profile indicatesa decreased likelihood of one or more of: a poor clinical outcome, goodclinical outcome, high risk of disease, low risk of disease, completeresponse, partial response, stable disease, non-response, andrecommended treatments for disease management. In some embodiments, adecrease in the quantitative score indicates an increased likelihood ofone or more of: a poor clinical outcome, good clinical outcome, highrisk of disease, low risk of disease, complete response, partialresponse, stable disease, non-response, and recommended treatments fordisease management.

In some applications, an increase in one or more microbes' thresholdvalues or quantitative score in a subject's microbiome profile indicatesan increased likelihood of one or more of: a poor clinical outcome, goodclinical outcome, high risk of disease, low risk of disease, completeresponse, partial response, stable disease, non-response, andrecommended treatments for disease management. In some applications, adecrease in one or more microbes' threshold values indicates anincreased likelihood of one or more of: a poor clinical outcome, goodclinical outcome, high risk of disease, low risk of disease, completeresponse, partial response, stable disease, non-response, andrecommended treatments for disease management.

In some applications, an increase in one or more microbes' thresholdvalues or quantitative score in a subject's microbiome profile indicatesa decreased likelihood of one or more of: a poor clinical outcome, goodclinical outcome, high risk of disease, low risk of disease, completeresponse, partial response, stable disease, non-response, andrecommended treatments for disease management. In some applications, adecrease in one or more microbes' threshold values indicates anincreased likelihood of one or more of: a poor clinical outcome, goodclinical outcome, high risk of disease, low risk of disease, completeresponse, partial response, stable disease, non-response, andrecommended treatments for disease management.

In some applications, a similar microbiome profile to a referenceprofile indicates an increased likelihood of one or more of: a poorclinical outcome, good clinical outcome, high risk of disease, low riskof disease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management. In someapplications, a dissimilar microbiome profile to a reference profileindicates one or more of: an increased likelihood of a poor clinicaloutcome, good clinical outcome, high risk of disease, low risk ofdisease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management.

In some applications, a similar microbiome profile to a referenceprofile indicates a decreased likelihood of one or more of: a poorclinical outcome, good clinical outcome, high risk of disease, low riskof disease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management. In someapplications, a dissimilar microbiome profile to a reference profileindicates one or more of: an increased likelihood of a poor clinicaloutcome, good clinical outcome, high risk of disease, low risk ofdisease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management.

In some applications, a dissimilar microbiome profile to a referenceprofile indicates an increased likelihood of one or more of: a poorclinical outcome, good clinical outcome, high risk of disease, low riskof disease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management. In someapplications, a dissimilar microbiome profile to a reference profileindicates one or more of: an increased likelihood of a poor clinicaloutcome, good clinical outcome, high risk of disease, low risk ofdisease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management.

In some applications, a dissimilar microbiome profile to a referenceprofile indicates a decreased likelihood of one or more of: a poorclinical outcome, good clinical outcome, high risk of disease, low riskof disease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management. In someapplications, a dissimilar microbiome profile to a reference profileindicates one or more of: an increased likelihood of a poor clinicaloutcome, good clinical outcome, high risk of disease, low risk ofdisease, complete response, partial response, stable disease,non-response, and recommended treatments for disease management.

An accurate 16S Copy Number can be required to accurately quantify using16S profiling. Using an incorrect database estimate of the copy numbercan be off by several factors, and in some cases an order of magnitudeor more.

FIG. 12 illustrates that de novo assembly for C. butyricum using methodsof the invention can result in the use of less contigs (e.g., 2 contigs)than those found in the database (e.g., 40 contigs).

FIG. 13(A) illustrates that de novo assembly using a method of theinvention can differentiate between several operon orderings and forthis example strain of C. butyricum a ‘type C’ ordering was discovered.(B) Tabulates the exact genomic coordinates for five of the butyratepathway genes for this strain.

Accuracy and Sensitivity

The methods provided herein can provide strain classification of agenera, species or sub-strain level of one or more microbes in a samplewith an accuracy of greater than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%,or 99.9%. The methods provided herein can provide strain quantificationof a genera, species or sub-strain level of one or more microbes in asample with an accuracy of greater than 1%, 20%, 30%, 40%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%,99.5%, 99.7%, or 99.9%.

The microbial profile can have an accuracy of 70% or greater based onmeasurement of 15 or fewer microbes in the biological sample. Suchprofiling method can have at least an accuracy greater than 70% based onmeasurement of no more than 2 microbes, 3 or fewer microbes, 4 or fewermicrobes, 5 or fewer microbes, 6 or fewer microbes, 7 or fewer microbes,8 or fewer microbes, 9 or fewer microbes, 10 or fewer microbes, 11 orfewer microbes, no more than 12 microbes, 13 or fewer microbes, 14 orfewer microbes, 15 or fewer microbes, 16 or fewer microbes, 18 or fewermicrobes, 19 or fewer microbes, 20 or fewer microbes, 25 or fewermicrobes, 30 or fewer microbes, 35 or fewer microbes, 40 or fewermicrobes, 45 or fewer microbes, 50 or fewer microbes, 55 or fewermicrobes, 60 or fewer microbes, 65 or fewer microbes, 70 or fewermicrobes, 75 or fewer microbes, 80 or fewer microbes, 85 or fewermicrobes, 90 or fewer microbes, or 100 or fewer microbes, 200 or fewermicrobes, 300 or fewer microbes, 400 or fewer microbes, 500 or fewermicrobes, 600 or fewer microbes, 700 or fewer microbes, or 800 or fewermicrobes.

The diagnostic methods provided by the present disclosure for thediseases provided herein can have at least one of a sensitivity of 70%or greater and specificity of greater than 70% based on measurement of15 or fewer microbes in the biological sample. Such diagnostic methodcan have at least one of a sensitivity greater than 70% and specificitygreater than 70% based on measurement of no more than 2 microbes, 3 orfewer microbes, 4 or fewer microbes, 5 or fewer microbes, 6 or fewermicrobes, 7 or fewer microbes, 8 or fewer microbes, 9 or fewer microbes,10 or fewer microbes, 11 or fewer microbes, no more than 12 microbes, 13or fewer microbes, 14 or fewer microbes, 15 or fewer microbes, 16 orfewer microbes, 18 or fewer microbes, 19 or fewer microbes, 20 or fewermicrobes, 25 or fewer microbes, 30 or fewer microbes, 35 or fewermicrobes, 40 or fewer microbes, 45 or fewer microbes, 50 or fewermicrobes, 55 or fewer microbes, 60 or fewer microbes, 65 or fewermicrobes, 70 or fewer microbes, 75 or fewer microbes, 80 or fewermicrobes, 85 or fewer microbes, 90 or fewer microbes, or 100 or fewermicrobes, 200 or fewer microbes, 300 or fewer microbes, 400 or fewermicrobes, 500 or fewer microbes, 600 or fewer microbes, 700 or fewermicrobes or 800 or fewer microbes.

The methods provided herein can provide a health status of a subjectwith a specificity greater than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%,or 99.9% receiver operating characteristic (ROC). The methods providedherein can provide a health status of a subject with a sensitivitylesser than 1%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.7%, or 99.9% ROC.

Methods for Treating a Subject

The disclosure provides methods for treating a subject. Altering thecomposition of a microbiome in a subject can have desired healthconsequences. Compositions of the disclosure can be administered as atherapeutic and/or a cosmetic for treating a health condition.Treatments designed to alter the host microbiome(s) can result in areduction of patient symptoms, prevention of disease, and or treatmentof the disease or health condition. For example, modification of the gutmicrobiome can reduce the risk for health conditions such as metabolicdisorders.

The disclosure provides methods for the restoration of a microbialhabitat of a subject to a healthy state. The method can comprisemicrobiome correction and/or adjustment including for example,replenishing native microbes, removing pathogenic microbes,administering prebiotics, and growth factors necessary for microbiomesurvival. In some embodiments, the method also comprises administeringantimicrobial agents such as antibiotics.

Based on the microbiome profile, the present disclosure provides methodsfor generalized-treatment recommendation for a subject as well asmethods for subject-specific treatment recommendation. Methods fortreatments can comprise one of the following steps: determining a firstratio of a level of a subject-specific microbiome profile to a level ofa second microbiome profile in a biological sample obtained from atleast one subject, detecting a presence or absence of a disease in thesubject based upon the determining, and recommending to the subject atleast one generalized or subject-specific treatment to amelioratedisease symptoms.

FIG. 1 depicts some non-limiting heath conditions that can be affectedby the microbiome. These health conditions can include, for example,Type 2 Diabetes Mellitus (T2DM), preterm labor, chronic fatiguesyndrome, skin conditions such as acne, allergies, autism, asthma,depression, hypertension, irritable bowel syndrome, metabolic syndrome,obesity, lactose intolerance, oral thrush, ulcerative colitis, drugmetabolism, vaginosis, atopic dermititus, psoriasis, Type I DiabetesMellitus (T1DM), Multiple Sclerosis, neurological disorders such asParkinson's disease, Clostridium Difficile infection, Inflammatory BowelDisease, Crohn's Disease, heart disease, diabetic foot ulcers,bacteremia, infantile colic, cancer, cystic fibrosis, multiplesclerosis, urinary tract infection, radiation enteropathy, drugmetabolism, dental cavities, and halitosis. The present disclosure canprovide for a diagnostic assay of at least one microbiome that includesa report that gives guidance on health status or treatment modalitiesfor the health conditions described herein. The present disclosure canalso provide therapeutic and/or cosmetic formulations for treatment ofhealth conditions described herein.

Inflammatory bowel disease (IBD) can involve chronic inflammation of allor part of the digestive tract. IBD can lead to ulcerative colitisand/or Crohn's disease. IBD can be painful and debilitating, andsometimes leads to life-threatening complications.

Preterm labor can occur when contractions begin to open the cervixbefore 37 weeks of pregnancy. The earlier premature birth happens, thegreater the health risks for the developing baby. Many premature babiesneed special care in the neonatal intensive care unit. Premature babiescan also have long-term mental and physical disabilities.

Obesity can be a complex disorder involving an excessive amount of bodyfat. Obesity can increase the risk of diseases and health problems suchas heart disease, diabetes and high blood pressure.

Peripheral neuropathy is the most common form of diabetic neuropathy. Inperipheral neuropathy, the feet and legs can be affected first, followedby the hands and arms. Possible signs and symptoms of peripheralneuropathy can include serious foot problems, such as ulcers,infections, deformities, and bone and joint pain.

Bacteremia or septicemia can refer to the presence of bacteria in theblood. A diagnosis of bacteremia can be confirmed by a blood culture.Treatment can require hospitalization and intravenous antibiotics.Bacteremia can quickly progress to severe sepsis.

Acne is a skin condition that can occur when the hair follicles becomeplugged with oil and dead skin cells. Acne can appear on the face, neck,chest, back and shoulders. Depending on the severity of the acne, thecondition can cause emotional distress and lead to scarring of the skin.

Infantile colic can refer to a condition involving an infant withexcessive crying, irritability, or fussiness. Babies with colic can cryfor more than three hours a day, three days a week for three weeks orlonger.

Type 2 diabetes also known as adult-onset or noninsulin-dependentdiabetes can be a chronic condition that affects the way the bodymetabolizes glucose. In type 2 diabetes, the body can either resist theeffects of insulin or not produce enough insulin to maintain a normalglucose level. Untreated, type 2 diabetes can be life-threatening.Symptoms of Type 2 diabetes can include, for example, increased thirstand frequent urination, increased hunger, weight gain, weight loss,fatigue, blurred vision, slow-healing sores or frequent infections,areas of darkened skin, and acanthosis nigricans.

Clostridium difficile also called C. difficile or C. diff is a bacteriumthat can cause symptoms ranging from diarrhea to life-threateninginflammation of the colon.

Asthma is a condition in which the airways can become narrow, swell andproduce extra mucus. The changes in the airway can make breathingdifficult and trigger coughing, wheezing and shortness of breath.

Autism spectrum disorder (ASD) can be a serious neurodevelopmentaldisorder that impairs a child's ability to communicate and interact withothers. The disorder also includes restricted repetitive behaviors,interests and activities. ASC can include autism, Asperger's syndrome,childhood disintegrative disorder and pervasive developmental disordernot otherwise specified.

Psoriasis is a persistent and chronic skin condition that can change thelife cycle of skin cells. Psoriasis can cause cells to build up rapidlyon the surface of the skin. The extra skin cells can form thick, silveryscales and itchy, dry, red patches that are sometimes painful.

Allergies can occur when the immune system reacts to a foreign substancesuch as pollen, bee venom or pet dander. The immune system's reaction toan allergen can involve inflammation of the skin, sinuses, airways ordigestive system.

Cardiovascular diseases can affect the heart, arteries and veins of thebody. Examples of some cardiovascular disease include heart valvedisease, coronary artery disease, congenital heart disease in adults andcongenital heart spontaneous coronary artery dissection, heart failure,heart rhythm disorders (arrhythmias).

Cancer can refer to any one of a large number of proliferative diseasescharacterized by the development of abnormal cells that divideuncontrollably and have the ability to infiltrate and destroy normalbody tissues and organs.

Depression, major depression, major depressive disorder or clinicaldepression can refer to a mood disorder that causes a persistent feelingof sadness and loss of interest. Depression can affect how a subjectfeels, thinks and behaves and can lead to a variety of emotional andphysical problems.

Cystic fibrosis is a life-threatening genetic disorder that can causesevere damage to the lungs and digestive system. Cystic fibrosis affectsthe cells that produce secreted fluids such as mucus, sweat anddigestive juices that act as lubricants in the body. These secretedfluids are normally thin and slippery but in cystic fibrosis thesecretions to become thick and sticky resulting in plugging up tubes,ducts and passageways, especially in the lungs and pancreas.

Multiple sclerosis is a disease in which the immune system attacks theprotective sheath (myelin) that covers the nerves. Myelin damagedisrupts communication between the brain and the rest of the body.Ultimately, the nerves themselves may deteriorate a process that'scurrently irreversible.

Urinary tract infection is an infection in any part of the urinarysystem (e.g. kidneys, ureters, bladder and urethra). Urinary tractinfection can be painful. Serious consequences can occur if a urinarytract infection spreads to the kidneys.

Radiation enteropathy can refer to radiation-induced gastrointestinalinjury. Radiotherapy is a mainstay of oncological treatment for avariety of malignant diseases. Radiotherapy can be administered to theabdomen and pelvis of patients with gastrointestinal (GI), urologicaland gynaecological cancers.

Drug metabolism can refer to the rate at which the body breaks down asdrug after administration.

Chronic fatigue syndrome is a complicated disorder characterized byextreme fatigue that cannot be explained by an underlying medicalcondition. The fatigue may worsen with physical or mental activity, butmay not improve with rest.

Type 1 diabetes, also known as juvenile diabetes or insulin-dependentdiabetes, is a chronic condition in which the pancreas produces littleor no insulin, a hormone needed to allow sugar (glucose) to enter cellsto produce energy. Various factors may contribute to type ldiabetes,including genetics and exposure to certain viruses. Although type 1diabetes typically appears during childhood or adolescence, it also candevelop in adults.

Dental cavities can be caused by the conversion of sugar from food toelongated, sticky sugar chains through a bacterially producedglucansucrase enzyme. One approach to treat and/or prevent cavities canbe to reduce the proportion of Streptococcus mutans, which is thebacteria associated with tooth decay. This approach can leave enzymessuch as enzymes used by the body to break down starches intact whilesimultaneously minimizing the rate of cavity formation.

Halitosis is a dental condition in which excessively bad breath can beproduced by the microbial flora present in a subject's mouth. Examplesof halitosis related microbes include gram-negative bacteria such asPrevotella intermedia, Porphyromonas gingivalis, Treponema denticola.Methods of the disclosure can be used to generate a list comprisingproblematic and protective microbes associated with halitosis.

Antibiotics can alter microbial populations in the gastrointestinaltract. This alteration can result in antimicrobial-associated diarrheaand/or colitis.

Obesity can occur in subjects with a body mass index of 30 or greater.Obesity can be associated with, for example, breathlessness, increasedsweating, snoring, difficulty sleeping, inability to cope with suddenphysical activity, fatigue, back pain, joint pain, high blood pressure,hypertension, high cholesterol levels, coronary heart disease, stroke,thirst, frequent urination, and diabetes.

A subject treated with the microbial compositions of the invention canlose weight. The subject can lose, for example, about: 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 pounds of body weight.

A subject treated with the microbial compositions of the invention canlose weight. The subject can lose, for example, about: 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 pounds of body weight.

A subject treated with the microbial compositions of the invention cangain weight. The subject can gain, for example, about: 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 pounds of body weight.

The body mass index of a subject treated with the microbial compositionsof the invention can be reduced to, for example, less than 30, betweenabout 25 to 30, or less than about 25.

Microbial compositions of the invention can increase blood glucoselevels. Microbial compositions of the invention can decrease bloodglucose levels. An oral glucose tolerance test (OGTT) can be used tomeasure glucose tolerance. Fasting plasma glucose, measured before theOGTT begins, of below 6.1 mmol/L (i.e. 110 mg/dL) can be considerednormal. Fasting levels between 6.1 and 7.0 mmol/L (i.e. 110 and 125mg/dL) can be considered borderline (e.g., impaired fasting glycaemia).Fasting levels repeatedly at or above 7.0 mmol/L (i.e. 126 mg/dL) can bediagnostic of diabetes. Microbial compositions of the invention candecrease blood glucose levels to normal levels, for example, below 6.1mmol/L (i.e. 110 mg/dL). Microbial compositions of the invention candecrease blood glucose levels to below diabetes levels or belowborderline levels as described herein.

Metabolic Diseases

Modifying a patient's microbiome, e.g. gut, intestinal tract, and/orcolon microbiome, can result in prevention and/or treatment of ametabolic health condition, including: T2DM, T1DM, obesity, metabolicdisorder, insulin resistance, and other diseases.

In some embodiments, the metabolic condition is T2DM. In someembodiments, the metabolic condition is obesity. In some embodiments,the metabolic condition is T1DM.

Butyrate is an anti-inflammatory factor that can affects gutpermeability. Low levels of butyrate producing bacteria (e.g.Clostridium clusters XIVa and IV) as well as reduced lactate producingbacteria (e.g. Bifidobacterium adolescentis) can be correlated withT1DM. Low levels of butyrate producing bacteria (e.g. Clostridiumclusters XIVa and IV) as well as reduced lactate producing bacteria(e.g. Bifidobacterium adolescentis) can be correlated with T2DM. Lowlevels of butyrate producing bacteria (e.g. Clostridium clusters XIVaand IV) as well as reduced lactate producing bacteria (e.g.Bifidobacterium adolescentis) can be correlated with obesity. Low levelsof butyrate producing bacteria (e.g. Clostridium clusters XIVa and IV)as well as reduced lactate producing bacteria (e.g. Bifidobacteriumadolescentis) can be correlated with a metabolic disorder. Subsets of aformulation that comprises at least one primary fermenter and at leastone secondary fermenter can be used for the treatment and/or mitigateprogression of a metabolic health condition, for example, T1DM.

FIG. 2 depicts a critical digestive pathway that can impactmetabolic-related health conditions. Alteration of the pathway usingmicrobial compositions of the invention can result in treatment. In thecolon, dietary fiber can be processed by butyrate-producingmicroorganisms to produce butyrate (i.e. butanoate), which is a shortchain fatty acid (SCFA). In turn, butyrate can initiate G-proteincoupled receptor (GPCR) signaling, leading to glucagon-like peptide-1(GLP-1) secretion which can result in increased insulin sensitivityand/or decreased appetite. By altering the butyrate-producing microbiomein a subject, e.g. with T2DM or insulin insensitivity, the pathway canbe stimulated. In some patients, insulin sensitivity can be increasedand/or restored to pre-diabetic levels with a microbial composition.

In some aspects of the invention, strains of interest are chosen byidentifying a superset of bacteria that play a role in the functionalpathway that leads to GLP-1 production (e.g. bacteria that have butyratekinase, butyrate coenzyme A (CoA), and/or butyrate CoA transferasegenes).

Butyrate kinase is an enzyme that can belong to a family oftransferases, for example those transferring phosphorus-containinggroups (e.g., phosphotransferases) with a carboxy group as acceptor. Thesystematic name of this enzyme class can be ATP:butanoate1-phosphotransferase. Butyrate kinase can participate in butyratemetabolism. Butyrate kinase can catalyze the following reaction:ADP+butyryl-phosphate

ATP+butyrate

Butyrate-Coenzyme A, also butyryl-coenzyme A, can be a coenzymeA-activated form of butyric acid. It can be acted upon by butyryl-CoAdehydrogenase and can be an intermediary compound inacetone-butanol-ethanol fermentation. Butyrate-Coenzyme A can beinvolved in butyrate metabolism.

Butyrate-Coenzyme A transferase, also known as butyrate-acetoacetateCoA-transferase, can belong to a family of transferases, for example,the CoA-transferases. The systematic name of this enzyme class can bebutanoyl-CoA:acetoacetate CoA-transferase. Other names in common use caninclude butyryl coenzyme A-acetoacetate coenzyme A-transferase, andbutyryl-CoA-acetoacetate CoA-transferase. Butyrate-Coenzyme Atransferase can catalyze the following chemical reaction:butanoyl-CoA+acetoacetate

butanoate+acetoacetyl-CoA

Butyryl-CoA dehydrogenase can belong to the family of oxidoreductases,for example, those acting on the CH—CH group of donor with otheracceptors. The systematic name of this enzyme class can bebutanoyl-CoA:acceptor 2,3-oxidoreductase. Other names in common use caninclude butyryl dehydrogenase, unsaturated acyl-CoA reductase, ethylenereductase, enoyl-coenzyme A reductase, unsaturated acyl coenzyme Areductase, butyryl coenzyme A dehydrogenase, short-chain acyl CoAdehydrogenase, short-chain acyl-coenzyme A dehydrogenase, 3-hydroxyacylCoA reductase, and butanoyl-CoA:(acceptor) 2,3-oxidoreductase.Non-limiting examples of metabolic pathways that butyryl-CoAdehydrogenase can participate in include: fatty acid metabolism; valine,leucine and isoleucine degradation; and butanoate metabolism.Butyryl-CoA dehydrogenase can employ one cofactor, FAD. Butyryl-CoAdehydrogenase can catalyze the following reaction:butyryl-CoA+acceptor

2-butenoyl-CoA+reduced acceptor

Beta-hydroxybutyryl- CoA dehydrogenase or 3-hydroxybutyryl-CoAdehydrogenase can belong to a family of oxidoreductases, for example,those acting on the CH—OH group of donor with NAD+ or NADP+as acceptor.The systematic name of the enzyme class can be(S)-3-hydroxybutanoyl-CoA:NADP+oxidoreductase. Other names in common usecan include beta-hydroxybutyryl coenzyme A dehydrogenase,L(+)-3-hydroxybutyryl-CoA dehydrogenase, BHBD, dehydrogenase,L-3-hydroxybutyryl coenzyme A (nicotinamide adenine, dinucleotidephosphate), L-(+)-3-hydroxybutyryl-CoA dehydrogenase, and3-hydroxybutyryl-CoA dehydrogenase. Beta-hydroxybutyryl- CoAdehydrogenase enzyme can participate in benzoate degradation via coaligation. Beta-hydroxybutyryl- CoA dehydrogenase enzyme can participatein butanoate metabolism. Beta-hydroxybutyryl- CoA dehydrogenase cancatalyze the following reaction:(S)-3-hydroxybutanoyl-CoA+NADP⁺

3-acetoacetyl-CoA+NADPH+H⁺

Crotonase can comprise enzymes with, for example, dehalogenase,hydratase, isomerase activities. Crotonase can be implicated incarbon-carbon bond formation, cleavage, and hydrolysis of thioesters.Enzymes in the crotonase superfamily can include, for example, enoyl-CoAhydratase which can catalyse the hydratation of 2-trans-enoyl-CoA into3-hydroxyacyl-CoA; 3-2trans-enoyl-CoA isomerase or dodecenoyl-CoAisomerise (e.g., EC 5.3.3.8), which can shift the 3-double bond of theintermediates of unsaturated fatty acid oxidation to the 2-transposition; 3-hydroxbutyryl-CoA dehydratase (e.g., crotonase; EC4.2.1.55), which can be involved in the butyrate/butanol-producingpathway; 4-Chlorobenzoyl-CoA dehalogenase (e.g., EC 3.8.1.6) which cancatalyze the conversion of 4-chlorobenzoate-CoA to4-hydroxybenzoate-CoA; dienoyl-CoA isomerase, which can catalyze theisomerisation of 3-trans,5-cis-dienoyl-CoA to2-trans,4-trans-dienoyl-CoA; naphthoate synthase (e.g., MenB, or DHNAsynthetase; EC 4.1.3.36), which can be involved in the biosynthesis ofmenaquinone (e.g., vitamin K2); carnitine racemase (e.g., gene caiD),which can catalyze the reversible conversion of crotonobetaine toL-carnitine in Escherichia coli; Methylmalonyl CoA decarboxylase (e.g.,MMCD; EC 4.1.1.41); carboxymethylproline synthase (e.g., CarB), whichcan be involved in carbapenem biosynthesis; 6-oxo camphor hydrolase,which can catalyze the desymmetrization of bicyclic beta-diketones tooptically active keto acids; the alpha subunit of fatty acid oxidationcomplex, a multi-enzyme complex that can catalyze the last threereactions in the fatty acid beta-oxidation cycle; and AUH protein, whichcan be a bifunctional RNA-binding homologue of enoyl-CoA hydratase.

Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT),can convert two units of acetyl-CoA to acetoacetyl CoA, for example, inthe mevalonate pathway. Thiolases can include, for example, degradativethiolases (e.g., EC 2.3.1.16) and biosynthetic thiolases (e.g., EC2.3.1.9). 3-ketoacyl-CoA thiolase, also called thiolase I, can beinvolved in degradative pathways such as fatty acid beta-oxidation.Acetoacetyl-CoA thiolase, also called thiolase II, can be specific forthe thiolysis of acetoacetyl-CoA and can be involved in biosyntheticpathways such as poly beta-hydroxybutyric acid synthesis or steroidbiogenesis. A thiolase can catalyze the following reaction:

As shown in FIG. 2, production of butyrate can involve two major phasesor microbes, for example, a primary fermenter and a secondary fermenter.The primary fermenter can produce intermediate molecules (e.g. lactate,acetate) when given an energy source (e.g. fiber). The secondaryfermenter can convert the intermediate molecules produced by the primaryfermenter into butyrate. Non-limiting examples of primary fermenterinclude Akkermansia muciniphila, Bifidobacterium adolescentis,Bifidobacterium infantis and Bifidobacterium longum. Non-limitingexamples of secondary fermenter include Clostridium beijerinckii,Clostridium butyricum, Clostridium indolis, Eubacterium hallii, andFaecalibacterium prausnitzii. A combination of primary and secondaryfermenters can be used to produce butyrate in a subject. Subsets of aformulation that comprises at least one primary fermenter and at leastone secondary fermenter can be used for the treatment and/or mitigateprogression of a metabolic health condition, for example, T2DM andobesity. The formulation can additionally comprise a prebiotic.

In some embodiments, a therapeutic composition comprises at least oneprimary fermenter and at least one secondary fermenter. In someembodiments, a therapeutic composition comprises at least one primaryfermenter, at least one secondary fermenter, and at least one prebiotic.In one non-limiting example, a therapeutic composition can compriseBifidobacterium adolescentis, Clostridium indolis, and inulin. Inanother non-limiting example, a therapeutic composition can compriseBifidobacterium longum, Faecalibacterium prausnitzii, and starch.

Akkermansia mucimphila can be a gram negative, strict anaerobe that canplay a role in mucin degradation. Levels of Akkermansia mucimphila canbe reduced in subjects with metabolic disorders, for example, obesityand T2DM. Akkermansia mucimphila can protect against metabolic disorder,for example, through increased levels of endocannabinoids that controlinflammation, the gut barrier, and gut peptide secretion. Akkermansiamucimphila can serve as a primary fermenter.

Bifidobacterium adolescentis can be a gram-positive anaerobe, which canbe found in healthy human gut from infancy. Bifidobacterium adolescentiscan synthesize B vitamins. Bifidobacterium adolescentis can serve as aprimary fermenter.

Bifidobacterium infantis can be a gram-positive, catalase negative,micro-aerotolerant anaerobe. Bifidobacterium infantis can serve as aprimary fermenter.

Bifidobacterium longum can be a gram-positive, catalase negative,micro-aerotolerant anaerobe. Bifidobacterium longum can serve as aprimary fermenter.

Clostridium beijerinckii can be a gram-positive, strict anaerobe thatbelongs to Clostridial cluster I. Clostridium beijerinckii can serve asa secondary fermenter.

Clostridium butyricum can be a gram-positive, strict anaerobe that canserve as a secondary fermenter.

Clostridium indolis can be a gram-positive, strict anaerobe that belongsto Clostridial cluster XIVA. Clostridium indolis can serve as asecondary fermenter.

Eubacterium hallii can be a gram-positive, anaerobe that belongs toArrangement A Clostridial cluster XIVA. Eubacterium hallii can serve asa secondary fermenter.

Faecalibacterium prausnitzii can be a gram-positive, anaerobe belongingto Clostridial cluster IV. Faecalibacterium prausnitzii can be one ofthe most common gut bacteria and the largest butyrate producer.Faecalibacterium prausnitzii can serve as a secondary fermenter.

Non-limiting examples of genes involved in the generation of butyrateinclude: butyryl-CoA dehydrogenase, beta-hydroxybutyryl- CoAdehydrogenase or 3-hydroxybutyryl-CoA dehydrogenase, crotonase, electrontransfer protein a, electron transfer protein b, and thiolase

Measuring the microbiome of hosts can show that microbiomes lackingvarious strains of microorganisms can result in a health conditionand/or disease state (e.g. T2DM and obesity). Restoring one or morelacking strains (e.g. via a bacterial strain such as E. hallii ortreatment with fermented milk products) can result in alteration of thehealth condition. Some non-limiting examples include altering the gutmicrobiome such that the host has an increased capacity for energyharvest, increased insulin sensitivity, and/or decreased appetite.

To treat metabolic conditions, for example, T2DM, obesity, and/or T1DM,one or more of the following microorganisms can be administered to thecolon: Butyrivibrio fibrisolvens, Clostridium acetobutylicum,Clostridium beijerinckii, Faecalibacterium prausnitzii, Roseburiacecicola, Clostridium butyricum, Bifidobacterium infantis,Bifidobacterium longum, Bifidobacterium adolescentis, Streptococcusmutans, Ruminococcus gnavus, Roseburia inulinivorans, Akkermansiamuciniphila, Fibrobacter succinogenes, Ruminococcus flavefaciens,Anaerostipes caccae, Eubacterium hallii, Clostridium indolis,Eubacterium rectale, or any combination thereof. The microorganisms canbe administered with a prebiotic.

In some embodiments, a pharmaceutical composition comprising one or moreof the following microorganisms are administered for the treatment ofmetabolic conditions: Butyrivibrio fibrisolvens, Clostridiumacetobutylicum, Clostridium beijerinckii, Faecalibacterium prausnitzii,Roseburia cecicola, Clostridium butyricum, Bifidobacterium infantis,Bifidobacterium longum, Bifidobacterium adolescentis, Streptococcusmutans, Ruminococcus gnavus, Roseburia inulinivorans, Akkermansiamuciniphila, Fibrobacter succinogenes, Ruminococcus flavefaciens,Anaerostipes caccae, Eubacterium hallii, Clostridium indolis andEubacterium rectale. The composition can additionally comprise aprebiotic.

FIG. 6 depicts an exemplary method to identify microorganism strains tobe used in the treatment of T2DM. A multi-tiered approach can be used toidentify one or more microorganism strains for use as a therapeutic.Candidate strains can be found in scientific literature and studies.Candidate strains can be found by analyzing healthy and unhealthy hosts.Candidate strains can be filtered and/or selected for the ability to beadministered to a patient (e.g. biosafety level, availability to bemanufactured, growth conditions). Finally, an in silico consortia can bedetermined.

In some embodiments, the prebiotic and probiotic consortia are chosen tocreate an entirely self-sufficient system that does not require anyexternal input. For example, a subject with T2DM can be treated with acombination of SCFA-producing probiotics and prebiotics comprisingdietary fiber and other agents required for the activity of theSCFA-producing probiotics. In this manner, the prebiotic and probioticform a self-sufficient system, wherein the probiotic converts theprebiotic dietary fiber to SCFAs (butyrate, acetate, propionate), whichcan trigger downstream signaling for controlling obesity, diabetes andpromote weigh loss in the subject.

Also provided are methods to generate probiotics against a subject'smicrobiome composition. The microbiome composition can have an effect onthe subject's disease status and clinical treatment response.Compositions of the disclosure can be tailored to suit the microbiomecomposition of a subject for effective treatment of symptoms associatedwith a health condition. For example, therapeutic formulations for obeseindividuals can differ from therapeutic formulations for non-obeseindividuals for the treatment of a specific disorder based ondifferences in their microbiota.

In some embodiments, methods for achieving weight loss by targetingrebalancing of the gut microbiome comprise: using specific probioticstrains, using appropriate prebiotics, diet recommendation, and periodicmonitoring. For example, the weight loss methods can comprise replacingprevotellas (a group within the Bacteroidetes phylum) and selenomonasfrom the microbiomes of overweight subjects with native probioticstrains from a healthy subject. Prebiotics can comprise dietary fiberand agents required for sustenance of the native probiotics.

Also provided are methods of formulating prebiotics and/or probioticcombinations to treat health conditions. A composition comprisingprebiotics and/or probiotics can prevent, for example, gastrointestinalinfections through production of antimicrobial factors, stimulation ofthe host immune system, and/or competition with pathogens for nutrientsor host binding sites. A combination of probiotics and prebiotics canprovide a complete system for producing amino acids, polyphenols,vitamins, and other compounds of nutritive value in a subject.

Microbe Compositions

In one aspect of the invention, a strain consortia comprising one ormore microorganisms selected from the group consisting of: Akkermansiamuciniphila, Anaerostipes caccae, Bifidobacterium adolescentis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum,Clostridium aminophilum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium colinum, Clostridium indolis, Clostridiumorbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacteriumrectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes,Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillusbulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospiraguilliermondii, Roseburia cecicola, Roseburia inulinivorans,Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum,Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis,Streptococcus mutans, Streptococcus thermophilus, Anaerofustisstercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis,Clostridium sporogenes, Clostridium tetani, Coprococcus, Coprococcuseutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacteriumventriosum, Roseburia faeccis, Roseburia hominis, Roseburiaintestinalis, and any combination thereof, can be used to treat ametabolic disorder such as obesity or T2DM.

A therapeutic consortium can comprise one or more microorganismsselected from the group consisting of: Akkermansia muciniphila,Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacteriumbifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibriofibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum,Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum,Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium,Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii,Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillusbrevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscaucasicus, Lactobacillus fermentum, Lactobacillus helveticus,Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri,Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburiacecicola, Roseburia inulinivorans, Ruminococcus flavefaciens,Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris,Streptococcus faecium, Streptococcus infantis, Streptococcus mutans,Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipeshadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridiumtetani, Coprococcus, Coprococcus eutactus, Eubacterium cylindroides,Eubacterium dolichum, Eubacterium ventriosum, Roseburia faeccis,Roseburia hominis, Roseburia intestinalis, and any combination thereof.

In one aspect of the invention, microbe compositions comprising one ormore microorganisms selected from the group consisting of: Akkermansiamuciniphila, Anaerostipes caccae, Bifidobacterium adolescentis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Butyrivibrio fibrisolvens, Clostridium acetobutylicum,Clostridium aminophilum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium colinum, Clostridium indolis, Clostridiumorbiscindens, Enterococcus faecium, Eubacterium hallii, Eubacteriumrectale, Faecalibacterium prausnitzii, Fibrobacter succinogenes,Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillusbulgaricus, Lactobacillus casei, Lactobacillus caucasicus, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Oscillospiraguilliermondii, Roseburia cecicola, Roseburia inulinivorans,Ruminococcus flavefaciens, Ruminococcus gnavus, Ruminococcus obeum,Streptococcus cremoris, Streptococcus faecium, Streptococcus infantis,Streptococcus mutans, Streptococcus thermophilus, Anaerofustisstercorihominis, Anaerostipes hadrus, Anaerotruncus colihominis,Clostridium sporogenes, Clostridium tetani, Coprococcus, Coprococcuseutactus, Eubacterium cylindroides, Eubacterium dolichum, Eubacteriumventriosum, Roseburia faeccis, Roseburia hominis, Roseburiaintestinalis, and any combination thereof, can be used to treat ametabolic disorder such as obesity or T2DM.

In some embodiments, provided are therapeutic compositions to treat ametabolic disorder comprising a purified microorganism populationconsisting of bacteria with at least about: 70%, 75%, 80%, 85%, 87%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%sequence identity to the 16SrRNA and/or 23S rRNA of a microorganismselected from the group consisting of: Akkermansia muciniphila,Anaerostipes caccae, Bifidobacterium adolescentis, Bifidobacteriumbifidum, Bifidobacterium infantis, Bifidobacterium longum, Butyrivibriofibrisolvens, Clostridium acetobutylicum, Clostridium aminophilum,Clostridium beijerinckii, Clostridium butyricum, Clostridium colinum,Clostridium indolis, Clostridium orbiscindens, Enterococcus faecium,Eubacterium hallii, Eubacterium rectale, Faecalibacterium prausnitzii,Fibrobacter succinogenes, Lactobacillus acidophilus, Lactobacillusbrevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscaucasicus, Lactobacillus fermentum, Lactobacillus helveticus,Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri,Lactobacillus rhamnosus, Oscillospira guilliermondii, Roseburiacecicola, Roseburia inulinivorans, Ruminococcus flavefaciens,Ruminococcus gnavus, Ruminococcus obeum, Streptococcus cremoris,Streptococcus faecium, Streptococcus infantis, Streptococcus mutans,Streptococcus thermophilus, Anaerofustis stercorihominis, Anaerostipeshadrus, Anaerotruncus colihominis, Clostridium sporogenes, Clostridiumtetani, Coprococcus, Coprococcus eutactus, Eubacterium cylindroides,Eubacterium dolichum, Eubacterium ventriosum, Roseburia faeccis,Roseburia hominis, Roseburia intestinalis, and any combination thereof.

In some embodiments, provided are therapeutic compositions to treat ametabolic disorder comprising an isolated and/or purified microorganismpopulation consisting of bacteria with at least about: 70%, 75%, 80%,85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or100% sequence identity to the 16SrRNA and/or 23S rRNA of a microorganismselected from the group consisting of: Akkermansia muciniphila,Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacteriumlongum, Clostridium beijerinckii, Clostridium butyricum, Clostridiumindolis, Eubacterium hallii, and any combination thereof.

In some embodiments, provided are therapeutic compositions to treat ametabolic disorder comprising an isolated and/or purified microorganismpopulation consisting of bacteria with at least about: 70%, 75%, 80%,85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or100% sequence identity to the 16SrRNA and/or 23S rRNA of a microorganismselected from the group consisting of: Akkermansia muciniphila,Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacteriumlongum, Clostridium beijerinckii, Clostridium butyricum, Clostridiumindolis, Eubacterium hallii, Faecalibacterium prausnitzii, and anycombination thereof.

In some embodiments, provided are therapeutic compositions to treat ametabolic disorder comprising an isolated and/or purified microorganismpopulation comprising bacteria selected from the group consisting of:Akkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacteriuminfantis, Bifidobacterium longum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium indolis, Eubacterium hallii, and any combinationthereof.

In some embodiments, provided are therapeutic compositions to treat ametabolic disorder comprising an isolated and/or purified microorganismpopulation comprising bacteria selected from the group consisting of:Akkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacteriuminfantis, Bifidobacterium longum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium indolis, Eubacterium hallii, Faecalibacteriumprausnitzii, and any combination thereof.

In some embodiments, a therapeutic consortium comprises Akkermansiamuciniphila, Bifidobacterium adolescentis, Bifidobacterium infantis,Bifidobacterium longum, Clostridium beijerinckii, Clostridium butyricum,Clostridium indolis, and Eubacterium hallii.

In some embodiments, a therapeutic consortium comprises Akkermansiamuciniphila, Bifidobacterium adolescentis, Bifidobacterium infantis,Bifidobacterium longum, Clostridium beijerinckii, Clostridium butyricum,Clostridium indolis, Eubacterium hallii, and Faecalibacteriumprausnitzii.

In some embodiments, a therapeutic consortium consists essentially ofAkkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacteriuminfantis, Bifidobacterium longum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium indolis, and Eubacterium hallii.

In some embodiments, a therapeutic consortium consists essentially ofAkkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacteriuminfantis, Bifidobacterium longum, Clostridium beijerinckii, Clostridiumbutyricum, Clostridium indolis, Eubacterium hallii, and Faecalibacteriumprausnitzii.

In one embodiment, a therapeutic composition to treat a metabolicdisorder comprises an isolated and/or purified microorganism populationconsisting of bacteria with at least about: 70%, 75%, 80%, 85%, 87%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%sequence identity to the 16SrRNA and/or 23S rRNA of Akkermansiamuciniphila.

In one embodiment, a therapeutic composition to treat a metabolicdisorder comprises an isolated and/or purified microorganism populationconsisting of bacteria with at least about: 70%, 75%, 80%, 85%, 87%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%sequence identity to the 16SrRNA and/or 23S rRNA of Anaerostipes caccae.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Bifidobacterium adolescentis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Bifidobacterium bifidum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Bifidobacterium infantis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Bifidobacterium longum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Butyrivibrio fibrisolvens.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium acetobutylicum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium aminophilum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium beijerinckii.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium butyricum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium colinum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium indolis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium orbiscindens.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Enterococcus faecium.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Eubacterium hallii.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Eubacterium rectale.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Faecalibacterium prausnitzii.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Fibrobacter succinogenes.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus acidophilus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus brevis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Lactobacillus bulgaricus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus casei.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Lactobacillus caucasicus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus fermentum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Lactobacillus helveticus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Lactobacillus lactis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus plantarum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus reuteri.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Lactobacillus rhamnosus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Oscillospira guilliermondii.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Roseburia cecicola.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Roseburia inulinivorans.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Ruminococcus flavefaciens.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Ruminococcus gnavus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Ruminococcus obeum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Streptococcus cremoris.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA Streptococcus faecium.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Streptococcus infantis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Streptococcus mutans.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Streptococcus thermophilus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Anaerofustis stercorihominis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Anaerostipes hadrus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Anaerotruncus colihominis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium sporogenes.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium sporogenes.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium tetani.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Clostridium tetani.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Coprococcus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Coprococcus eutactus.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Eubacterium cylindroides.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Eubacterium dolichum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Eubacterium ventriosum.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Roseburia faeccis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or23S rRNA of Roseburia hominis.

In one embodiment, a therapeutic composition comprises an isolatedand/or purified microorganism population consisting of bacteria with atleast about: 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 100% sequence identity to the 16SrRNA and/or 23S rRNA of Roseburia intestinalis.

A therapeutic composition may comprise at least 1, at least 2, at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 45, or atleast 50, or at least 75, or at least 100 types of bacteria. Atherapeutic composition may comprise at most 1, at most 2, at most 3, atmost 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most10, at most 11, at most 12, at most 13, at most 14, at most 15, at most16, at most 17, at most 18, at most 19, at most 20, at most 21, at most22, at most 23, at most 24, at most 25, at most 26, at most 27, at most28, at most 29, at most 30, at most 31, at most 32, at most 33, at most34, at most 35, at most 36, at most 37, at most 38, at most 39, at most40, at most 45, or at most 50, or at most 75, or at most 100 types ofbacteria.

In some embodiments, combining one or more microbes in a therapeuticcomposition or consortia increases or maintains the stability of themicrobes in the composition compared with the stability of the microbesalone as illustrated in FIG. 9. A therapeutic consortium of microbes canprovide a synergistic stability compared with the individual strains.

In some embodiments, combining one or more microbes in a therapeuticcomposition or consortia can provide a synergistic effect whenadministered to the individual. For example, administration of a firstmicrobe may be beneficial to a subject and administration of a secondmicrobe may be beneficial to a subject but when the two microbes areadministered together to a subject, the benefit is greater than theeither benefit alone.

Different types of microbes in a therapeutic composition can be presentin the same amount or in different amounts. For example, the ratio oftwo bacteria in a therapeutic composition can be about 1:1, 1:2, 1:5,1:10, 1:25, 1:50, 1:100, 1:1000, 1:10,000, or 1:100,000.

In some embodiments, a therapeutic composition comprises at least oneprimary fermenter and at least one secondary fermenter. In someembodiments, a therapeutic composition comprises at least one primaryfermenter, at least one secondary fermenter, and at least one prebiotic.In one example, a therapeutic composition can comprise Clostridiumindolis, Bifidobacterium adolescentis, and inulin. In another example, atherapeutic composition can comprise Faecalibacterium prausnitzii,Bifidobacterium longum, and starch.

Microorganisms of the invention can be produced in any suitable mediumfor growth, some non-limiting examples include: RCM, GYT veg, BHI,PYGveg, nutrient media, minimal media, selective media, differentialmedia, and transport media. The growth medium can comprise a tracemineral. The growth medium can comprise a salt. The growth medium cancomprise a vitamin. The growth medium can comprise a buffer. The pH of agrowth medium can be, for example, about 7. The pH of a growth mediumcan be, for example, about 3, about, 4, about, 5, about 6, about 7, orabout 8. The growth medium can improve the maximum density a microbialstrain can grow to. The growth medium can allow for higher strainconcentrations. The growth medium can buffer acid production by amicrobial strain, which can minimize the inhibitory effect of, forexample, very low pH.

Table 1 shows trace minerals that can be added to a growth media:

TABLE 1 Trace minerals Trace minerals mg/L component medium CoCl₂ 0.65CuCl₂*2H₂O 0.03 H₃BO₃ 3.52 FeSO₄*7H₂O 1.50 MnCl₂*4H₂O 0.26 Na₂EDTA 25.01Na₂MoO₄*2H₂O 0.80 Na₂SeO₃ 0.39 NiCl₂ 0.65 ZnSO₄*7H₂O 0.29

Table 2 shows vitamins that can be added to a growth media. Theconcentrations shown in Table 2 can be final concentrations in thegrowth media.

TABLE 2 Vitamin solution. Vitamin Solution component mg/L mediumD-biotin 0.2 Ca-pantothenate 2.5 myoinositol 20 p-aminobenzoic acid 0.5pyridoxine 5 hydrochloride riboflavine 0.5 thiamine dichloride 10vitamin B12 0.2 nicotinic acid 5

Table 3 shows an illustrative growth medium:

TABLE 3 Illustrative growth medium recipe GYTveg broth (per liter):Component Amount Glucose 10 g HiVeg Hydrolysate 5 g Yeast Extract 10 gNa-thioglycolate 0.5 g Resazurin 80 μl (of 14 g/l stock) Vitaminsolution 10 ml Agar (for solid medium) 18 g

Table 4 shows an illustrative growth medium.

TABLE 4 Illustrative growth medium recipe GYTveg + CaCO₃ (per liter):Component Amount Glucose 10 g HiVeg Hydrolysate 5 g Yeast Extract 10 gNa-thioglycolate 0.5 g Resazurin 80 μl (of 14 g/l stock) Vitaminsolution 10 ml CaCO₃ 20 g Agar (for solid medium) 18 g

Table 5 shows an illustrative growth medium.

TABLE 5 Illustrative growth medium recipe PYGveg Component Amount perliter Glucose 5 g K₂HPO₄ 2 g Tween 80 1 ml Cystein-HCl 0.5 g Yeastextract 10 g HiVeg Extract 5 g HiVeg Peptone #1 5 g HiVeg Peptone #3 5 gVitamin Mix 100x 10 ml Salt solution 40 ml

Table 6 shows illustrative salts that can be added to a growth medium.The concentrations shown in Table 6 can be final concentrations in thegrowth medium.

TABLE 6 Salt solution Salt solution Component grams per liter CaCl₂ 2H₂O0.02 MgSO₄ 7H₂O 0.02 K₂HPO₄ 0.04 KH₂PO₄ 0.04 NaHCO₃ 0.4 NaCl 0.08

In some embodiments, the growth medium comprises PYGveg (e.g., Table 5),vitamins (e.g., Table 2), salt (e.g., Table 6), and a buffer.

Pharmaceutical Formulations

Provided herein are compositions that may be administered astherapeutics and/or cosmetics. One or more microorganisms describedherein can be used to create a pharmaceutical formulation comprising aneffective amount of the composition for treating a subject. Themicroorganisms can be in any formulation known in the art. Somenon-limiting examples can include topical, capsule, pill, enema, liquid,injection, and the like. In some embodiments, the one or more strainsdisclosed herein may be included in a food or beverage product,cosmetic, or nutritional supplement.

The formulation can include one or more active ingredients. Activeingredients can be selected from the group consisting of: antibiotics,prebiotics, probiotics, glycans (e.g., as decoys that would limitspecific bacterial/viral binding to the intestinal wall),bacteriophages, microorganisms and the like.

In some embodiments, the formulation comprises a prebiotic. In someembodiments, the prebiotic is inulin. The inulin can serve as an energysource for the microbial formulation.

A formulation can be administered by a suitable method for delivery toany part of the gastrointestinal tract of a subject including oralcavity, mouth, esophagus, stomach, duodenum, small intestine regionsincluding duodenum, jejunum, ileum, and large intestine regionsincluding cecum, colon, rectum, and anal canal. In some embodiments, thecomposition is formulated for delivery to the ileum and/or colon regionsof the gastrointestinal tract.

In some embodiments, administration of a formulation occurs orally, forexample, through a capsule, pill, powder, tablet, gel, or liquid,designed to release the composition in the gastrointestinal tract. Insome embodiments, administration of a formulation occurs by injection,for example, for a formulation comprising butyrate, propionate, acetate,and short-chain fatty acids. In some embodiments, the administration ofa formulation occurs by application to the skin, for example, cream,liquid, or patch. In some embodiments, administration of a formulationoccurs by a suppository and/or by enema. In some embodiments, acombination of administration routes is utilized.

Microbial compositions can be formulated as a dietary supplement.Microbial compositions can be incorporated with vitamin supplements.Microbial compositions can be formulated in a chewable form such as aprobiotic gummy. Microbial compositions can be incorporated into a formof food and/or drink. Non-limiting examples of food and drinks where themicrobial compositions can be incorporated include, for example, bars,shakes, juices, infant formula, beverages, frozen food products,fermented food products, and cultured dairy products such as yogurt,yogurt drink, cheese, acidophilus drinks, and kefir.

A formulation of the disclosure can be administered as part of a fecaltransplant process. A formulation can be administered to a subject by atube, for example, nasogastric tube, nasojejunal tube, nasoduodenaltube, oral gastric tube, oral jejunal tube, or oral duodenal tube. Aformulation can be administered to a subject by colonoscopy, endoscopy,sigmoidoscopy, and/or enema.

In some embodiments, the microbial composition is formulated such thatthe one or more microbes can replicate once they are delivered to thetarget habitat (e.g. the gut). In one non-limiting example, themicrobial composition is formulated in a pill, such that the pill has ashelf life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.In another non-limiting example, the storage of the microbialcomposition is formulated so that the microbes can reproduce once theyare in the gut. In some embodiments, other components may be added toaid in the shelf life of the microbial composition. In some embodiments,one or more microbes may be formulated in a manner that it is able tosurvive in a non-natural environment. For example, a microbe that isnative to the gut may not survive in an oxygen-rich environment. Toovercome this limitation, the microbe may be formulated in a pill thatcan reduce or eliminate the exposure to oxygen. Other strategies toenhance the shelf-life of microbes may include other microbes (e.g. ifthe bacterial consortia comprises a composition whereby one or morestrains is helpful for the survival of one or more strains).

In some embodiments, a microbial composition is lyophilized (e.g.,freeze-dried) and formulated as a powder, tablet, enteric-coated capsule(e.g. for delivery to ileum/colon), or pill that can be administered toa subject by any suitable route. The lyophilized formulation can bemixed with a saline or other solution prior to administration.

In some embodiments, a microbial composition is formulated for oraladministration, for example, as an enteric-coated capsule or pill, fordelivery of the contents of the formulation to the ileum and/or colonregions of a subject.

In some embodiments, the microbial composition is formulated for oraladministration. In some embodiments, the microbial composition isformulated as an enteric-coated pill or capsule for oral administration.In some embodiments, the microbial composition is formulated fordelivery of the microbes to the ileum region of a subject. In someembodiments, the microbial composition is formulated for delivery of themicrobes to the colon region (e.g. upper colon) of a subject. In someembodiments, the microbial composition is formulated for delivery of themicrobes to the ileum and colon regions of a subject.

An enteric-coating can protect the contents of the oral formulation, forexample, pill or capsule, from the acidity of the stomach and providedelivery to the ileum and/or upper colon regions. Non-limiting examplesof enteric coatings include pH sensitive polymers (e.g., eudragitFS30D), methyl acrylate-methacrylic acid copolymers, cellulose acetatesuccinate, hydroxy propyl methyl cellulose phthalate, hydroxy propylmethyl cellulose acetate succinate (e.g., hypromellose acetatesuccinate), polyvinyl acetate phthalate (PVAP), methylmethacrylate-methacrylic acid copolymers, shellac, cellulose acetatetrimellitate, sodium alginate, zein, other polymers, fatty acids, waxes,shellac, plastics, and plant fibers. In some embodiments, the entericcoating is formed by a pH sensitive polymer. In some embodiments, theenteric coating is formed by eudragit FS30D.

The enteric coating can be designed to dissolve at any suitable pH. Insome embodiments, the enteric coating is designed to dissolve at a pHgreater than about pH 6.5 to about pH 7.0. In some embodiments, theenteric coating is designed to dissolve at a pH greater than about pH6.5. In some embodiments, the enteric coating is designed to dissolve ata pH greater than about pH 7.0. The enteric coating can be designed todissolve at a pH greater than about: 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1,7.2, 7.3, 7.4, or 7.5 pH units.

In some embodiments, the administration of a formulation of thedisclosure can be preceded by, for example, colon cleansing methods suchas colon irrigation/hydrotherapy, enema, administration of laxatives,dietary supplements, dietary fiber, enzymes, and magnesium.

In some embodiments, the microbes are formulated as a population ofspores. Spore-containing formulations can be administered by anysuitable route described herein. Orally administered spore-containingformulations can survive the low pH environment of the stomach. Theamount of spores employed can be, for example, from about 1% w/w toabout 99% w/w of the entire formulation.

Formulations provded herein can include the addition of one or moreagents to the therapeutics or cosmetics in order to enhance stabilityand/or survival of the microbial formulation. Non-limiting example ofstabilizing agents include genetic elements, glycerin, ascorbic acid,skim milk, lactose, tween, alginate, xanthan gum, carrageenan gum,mannitol, palm oil, and poly-L-lysine (POPL).

In some embodiments, a formulation comprises one or more recombinantmicrobes or microbes that have been geneticallly modified. In otherembodiments, one or more microbes are not modified or recombinant. Insome embodiments, the formulation comprises microbes that can beregulated, for example, a microbe comprising an operon or promoter tocontrol microbial growth. Microbes of the invention can be produced,grown, or modified using any suitable methods, including recombinantmethods.

A formulation can be customized for a subject. A custom formulation cancomprise, for example, a prebiotic, a probiotic, an antibiotic, or acombination of active agents described herein. Data specific to thesubject comprising for example age, gender, and weight can be combinedwith an analysis result to provide a therapeutic agent customized to thesubject. For example, a subject's microbiome found to be low in aspecific microbe relative to a sub-population of healthy subjectsmatched for age and gender can be provided with a therapeutic and/orcosmetic formulation comprising the specific microbe to match that ofthe sub-population of healthy subjects having the same age and gender asthe subject.

In some embodiments, a formulation is administered before, during,and/or after treatment with an antimicrobial agent such as anantibiotic. For example, the formulation can be administered at leastabout 1 hour, 2 hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2weeks, 1 month, 6 months, or 1 year before and/or after treatment withan antibiotic. The formulation can be administered at most 1 hour, 2hours, 5 hours, 12 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 6months, or 1 year before and/or after treatment with an antibiotic.

In some embodiments, the formulation is administered after treatmentwith an antibiotic. For example, the formulation can be administeredafter the entire antibiotic regimen or course is complete.

In some embodiments, a formulation is administered before, during,and/or after food intake by a subject. In some embodiments, theformulation is administered with food intake by the subject. In someembodiments, the formulation is administered with (e.g., simultaneously)with food intake.

In some embodiments, the formulation is administered before food intakeby a subject. In some embodiments, the formulation is more effective orpotent at treating a microbial condition when administered before foodintake. For example, the formulation can be administered about 1 minute,about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes,about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 12 hours, or about 1 day before food intake by a subject. Forexample, the formulation can be administered at least about 1 minute,about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes,about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 12 hours, or about 1 day before food intake by a subject. Forexample, the formulation can be administered at most about 1 minute,about 2 minutes, about 3 minutes, about 5 minutes, about 10 minutes,about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 12 hours, or about 1 day before food intake by a subject.

In some embodiments, the formulation is administered after food intakeby the subject. In some embodiments, the formulation is more effectiveor potent at treating a microbial condition when administered after foodintake. For example, the formulation can be administered at least about1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 12hours, or 1 day after food intake by a subject. For example, theformulation can be administered at most about 1 minute, 2 minutes, 3minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 3 hours, 5 hours, 10 hours, 12 hours, or 1 day after foodintake by a subject.

Formulations provded herein can include those suitable for oralincluding buccal and sub-lingual, intranasal, topical, transdermal,transdermal patch, pulmonary, vaginal, rectal, suppository, mucosal,systemic, or parenteral including intramuscular, intraarterial,intrathecal, intradermal, intraperitoneal, subcutaneous, and intravenousadministration or in a form suitable for administration byaerosolization, inhalation or insufflation.

A therapeutic or cosmetic composition can include carriers andexcipients (including but not limited to buffers, carbohydrates, lipids,mannitol, proteins, polypeptides or amino acids such as glycine,antioxidants, bacteriostats, chelating agents, suspending agents,thickening agents and/or preservatives), metals (e.g., iron, calcium),salts, vitamins, minerals, water, oils including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like, saline solutions, aqueous dextroseand glycerol solutions, flavoring agents, coloring agents, detackifiersand other acceptable additives, adjuvants, or binders, otherpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH buffering agents,tonicity adjusting agents, emulsifying agents, wetting agents and thelike. Examples of excipients include starch, glucose, lactose, sucrose,gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like.

Non-limiting examples of pharmaceutically-acceptable excipients suitablefor use in the disclosure include granulating agents, binding agents,lubricating agents, disintegrating agents, sweetening agents, glidants,anti-adherents, anti-static agents, surfactants, anti-oxidants, gums,coating agents, coloring agents, flavouring agents, dispersion enhancer,disintegrant, coating agents, plasticizers, preservatives, suspendingagents, emulsifying agents, plant cellulosic material and spheronizationagents, and any combination thereof.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference in itsentirety.

A therapeutic or cosmetic composition can be substantially free ofpreservatives. In some applications, the compositon may contain at leastone preservative.

A therapeutic or cosmetic composition can be encapsulated within asuitable vehicle, for example, a liposome, a microspheres, or amicroparticle. Microspheres formed of polymers or proteins can betailored for passage through the gastrointestinal tract directly intothe blood stream. Alternatively, the compound can be incorporated andthe microspheres, or composite of microspheres, and implanted for slowrelease over a period of time ranging from days to months.

A therapeutic or cosmetic composition can be formulated as a sterilesolution or suspension. The therapeutic or cosmetic compositions can besterilized by conventional techniques or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized. The lyophilized preparation of the microbial compositioncan be packaged in a suitable form for oral administration, for example,capsule or pill.

The compositions can be administered topically and can be formulatedinto a variety of topically administrable compositions, such assolutions, suspensions, lotions, gels, pastes, medicated sticks, balms,creams, and ointments. Such pharmaceutical compositions can containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

The compositions can also be formulated in rectal compositions such asenemas, rectal gels, rectal foams, rectal aerosols, suppositories, jellysuppositories, or retention enemas, containing conventional suppositorybases such as cocoa butter or other glycerides, as well as syntheticpolymers such as polyvinylpyrrolidone, PEG, and the like. In suppositoryforms of the compositions, a low-melting wax such as a mixture of fattyacid glycerides, optionally in combination with cocoa butter, can beused.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the microbial compositionsdescribed herein are administered in pharmaceutical compositions to asubject having a disease or condition to be treated. In someembodiments, the subject is a mammal such as a human. Atherapeutically-effective amount can vary widely depending on theseverity of the disease, the age and relative health of the subject,potency of the formulation, and other factors. Subjects can be, forexample, humans, elderly adults, adults, adolescents, pre-adolescents,children, toddlers, infants, or neonates. A subject can be a patient. Asubject can be an individual enrolled in a clinical study. A subject canbe a laboratory animcal, for example, a mammal, or a rodent.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the microorganisms intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions described herein can be manufactured in aconventional manner, for example, by means of conventional mixing,dissolving, granulating, vitrification, spray-drying, lyophilizing,dragee-making, levigating, encapsulating, entrapping, emulsifying orcompression processes.

In some embodiments, the pharmaceutical composition is manufactured in adry form, for example, by spray-drying or lyophilization. In someembodiments, the formulation is prepared as a liquid capsule to maintainthe liquid form of the microbes.

Compositions provided herein can be stored at any suitable temperature.The formulation can be stored in cold storage, for example, at atemperature of about −80° C., about −20° C., about −4° C., or about 4°C. The storage temperature can be, for example, about 0° C., about 1°C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C.,about 7° C., about 8° C., about 9° C., about 10° C., about 12° C., about14° C., about 16° C., about 20° C., about 22° C., or about 25° C. Insome embodiments, the storage temperature is between about 2° C. toabout 8° C. Storage of microbial compositions at low temperatures, forexample from about 2° C. to about 8° C., can keep the microbes alive andincrease the efficiency of the composition, for example, when present ina liquid or gel formulation. Storage at freezing temperature, below 0°C., with a cryoprotectant can further extend stability.

The pH of the composition can range from about 3 to about 12. The pH ofthe composition can be, for example, from about 3 to about 4, from about4 to about 5, from about 5 to about 6, from about 6 to about 7, fromabout 7 to about 8, from about 8 to about 9, from about 9 to about 10,from about 10 to about 11, or from about 11 to about 12 pH units. The pHof the composition can be, for example, about 3, about 4, about 5, about6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units.The pH of the composition can be, for example, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11 or at least 12 pH units. The pH of the composition can be,for example, at most 3, at most 4, at most 5, at most 6, at most 7, atmost 8, at most 9, at most 10, at most 11, or at most 12 pH units. Ifthe pH is outside the range desired by the formulator, the pH can beadjusted by using sufficient pharmaceutically-acceptable acids andbases. In some embodiments, the pH of the composition is between about 4and about 6.

Pharmaceutical compositions containing microbes described herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions can be administered to asubject already suffering from a disease or condition, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease or condition, or to cure, heal, improve, or ameliorate thecondition. Microbial compositions can also be administered to lessen alikelihood of developing, contracting, or worsening a condition. Amountseffective for this use can vary based on the severity and course of thedisease or condition, previous therapy, the subject's health status,weight, and response to the drugs, and the judgment of the treatingphysician.

Multiple therapeutic agents can be administered in any order orsimultaneously. If simultaneously, the multiple therapeutic agents canbe provided in a single, unified form, or in multiple forms, forexample, as multiple separate pills. The composition can be packedtogether or separately, in a single package or in a plurality ofpackages. One or all of the therapeutic agents can be given in multipledoses. If not simultaneous, the timing between the multiple doses mayvary to as much as about a month.

Compositions described herein can be administered before, during, orafter the occurrence of a disease or condition, and the timing ofadministering the composition can vary. For example, the microbialcomposition can be used as a prophylactic and can be administeredcontinuously to subjects with a propensity to conditions or diseases inorder to lessen a likelihood of the occurrence of the disease orcondition. The microbial compositions can be administered to a subjectduring or as soon as possible after the onset of the symptoms. Theadministration of the microbial compositions can be initiated within thefirst 48 hours of the onset of the symptoms, within the first 24 hoursof the onset of the symptoms, within the first 6 hours of the onset ofthe symptoms, or within 3 hours of the onset of the symptoms. Theinitial administration can be via any route practical, such as by anyroute described herein using any formulation described herein. Amicrobial composition can be administered as soon as is practicableafter the onset of a disease or condition is detected or suspected, andfor a length of time necessary for the treatment of the disease, suchas, for example, from about 1 month to about 3 months. The length oftreatment can vary for each subject.

Compositions of the invention can be administered in combination withanother therapy, for example, immunotherapy, chemotherapy, radiotherapy,anti-inflammatory agents, anti-viral agents, anti-microbial agents, andanti-fungal agents.

Compositions of the invention can be packaged as a kit. In someembodiments, a kit includes written instructions on theadministration/use of the composition. The written material can be, forexample, a label. The written material can suggest conditions methods ofadministration. The instructions provide the subject and the supervisingphysician with the best guidance for achieving the optimal clinicaloutcome from the administration of the therapy. The written material canbe a label. In some embodiments, the label can be approved by aregulatory agency, for example the U.S. Food and Drug Administration(FDA), the European Medicines Agency (EMA), or other regulatoryagencies.

Dosing

The appropriate quantity of a therapeutic or cosmetic composition to beadministered, the number of treatments, and unit dose can vary accordingto a subject and/or the disease state of the subject.

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation can be divided into unit doses containingappropriate quantities of one or more microbial compositions. The unitdosage can be in the form of a package containing discrete quantities ofthe formulation. Non-limiting examples are liquids in vials or ampoules.Aqueous suspension compositions can be packaged in single-dosenon-reclosable containers. The composition can be in a multi-doseformat. Multiple-dose reclosable containers can be used, for example, incombination with a preservative. Formulations for parenteral injectioncan be presented in unit dosage form, for example, in ampoules, or inmulti-dose containers with a preservative.

The dosage can be in the form of a solid, semi-solid, or liquidcomposition. Non-limiting examples of dosage forms suitable for use inthe invention include feed, food, pellet, lozenge, liquid, elixir,aerosol, inhalant, spray, powder, tablet, pill, capsule, gel, geltab,nanosuspension, nanoparticle, microgel, suppository troches, aqueous oroily suspensions, ointment, patch, lotion, dentifrice, emulsion, creams,drops, dispersible powders or granules, emulsion in hard or soft gelcapsules, syrups, phytoceuticals, nutraceuticals, dietary supplement,and any combination thereof.

A microbe can be present in any suitable concentration in apharmaceutical composition. The concentration of a microbe can be forexample, from about 10¹to about 10¹⁸ colony forming units (CFU). Theconcentration of a microbe can be, for example, at least 10¹, at least10², at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, at least10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰, at least 10¹¹, at least10¹², at least 10¹³, at least 10¹⁴, at least 10¹⁵, at least 10¹⁶, atleast 10¹⁷, or at least 10¹⁸ CFU. The concentration of a microbe can be,for example, at most 10¹, at most 10², at most 10³, at most 10⁴, at most10⁵, at most 10⁶, at most 10⁷, at most 10⁸, at most 10⁹, at most 10¹⁰,at most 10¹¹, at most 10¹², at most 10¹³, at most 10¹⁴, at most 10¹⁵, atmost 10¹⁶, at most 10¹⁷, or at most 10¹⁸ CFU. In some embodiments, theconcentration of a microbe is from about 10⁸ CFU to about 10⁹ CFU. Insome embodiments, the concentration of a microbe is about 10⁸ CFU. Insome embodiments, the concentration of a microbe is about 10⁹ CFU.

Pharmaceutical compositions of the invention can be formulated with anysuitable therapeutically-effective concentration of prebiotic. Forexample, the therapeutically-effective concentration of a prebiotic canbe at least about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml,about 5 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml,about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml,about 95 mg/ml, about 100 mg/ml, about 110 mg/ml, about 125 mg/ml, about130 mg/ml, about 140 mg/ml, or about 150 mg/ml. For example, thetherapeutically-effective concentration of a prebiotic can be at mostabout 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml,about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml,about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95mg/ml, about 100 mg/ml, about 110 mg/ml, about 125 mg/ml, about 130mg/ml, about 140 mg/ml, or about 150 mg/ml. For example, thetherapeutically-effective concentration of a prebiotic can be about 1mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml,about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95 mg/ml,about 100 mg/ml, about 110 mg/ml, about 125 mg/ml, about 130 mg/ml,about 140 mg/ml, or about 150 mg/ml. In some embodiments, theconcentration of a prebiotic in a pharmaceutical composition is about 70mg/ml. In some embodiments, the prebiotic is inulin.

Pharmaceutical compositions of the invention can be administered, forexample, 1, 2, 3, 4, 5, or more times daily. Pharmaceutical compositionsof the invention can be administered, for example, daily, every otherday, three times a week, twice a week, once a week, or at otherappropriate intervals for treatment of the condition.

Computer Systems

The invention also provides a computer system that is configured toimplement the methods of the disclosure. The system can include acomputer server (“server”) that is programmed to implement the methodsdescribed herein. FIG. 7 depicts a system 700 adapted to enable a userto detect, analyze, and process data (e.g. sequencing data; strainclassification, functional pathways, epigenetic changes, patientinformation, external data, databases, microbiome strains; therapeuticconsortia, etc.). The system 700 includes a central computer server 701that is programmed to implement exemplary methods described herein. Theserver 701 includes a central processing unit (CPU, also “processor”)705 which can be a single core processor, a multi core processor, orplurality of processors for parallel processing, or cloud processors.The server 701 also includes memory 710 (e.g. random access memory,read-only memory, flash memory); electronic storage unit 715 (e.g. harddisk); communications interface 720 (e.g. network adaptor) forcommunicating with one or more other systems; and peripheral device 725which may include cache, other memory, data storage, and/or electronicdisplay adaptors. The memory 710, storage unit 715, interface 720, andperipheral device 725 are in communication with the processor 705through a communications bus (solid lines), such as a motherboard. Thestorage unit 715 can be a data storage unit for storing data. The server701 is operatively coupled to a computer network (“network”) 730 withthe aid of the communications interface 720. The network 730 can be theInternet, an intranet and/or an extranet, an intranet and/or extranetthat is in communication with the Internet, a telecommunication or datanetwork. The network 730 in some cases, with the aid of the server 701,can implement a peer-to-peer network, which may enable devices coupledto the server 701 to behave as a client or a server. Peripheral devicescan include, e.g. sequencers 725 or remote computer systems 740.

The storage unit 715 can store files, (e.g. any aspect of dataassociated with the invention). In some instances cloud storage is used.Cloud storage can be a model of data storage where the digital data isstored in logical pools, wherein the physical storage can span multipleservers and, in some instances, one or more locations. In someembodiments, the physical environment is owned and managed by a hostingcompany. Cloud storage services may be accessed, e.g., through aco-located cloud compute service, a web service application programminginterface (API) or by applications that utilize the API, such as clouddesktop storage, a cloud storage gateway or Web-based content managementsystems.

The server can communicate with one or more remote computer systemsthrough the network 730. The one or more remote computer systems may be,for example, personal computers, laptops, tablets, telephones, Smartphones, or personal digital assistants.

In some situations the system 700 includes a single server 701. In othersituations, the system includes multiple servers in communication withone another through an intranet, extranet and/or the Internet.

The server 701 can be adapted to store information. Such information canbe stored on the storage unit 715 or the server 701 and such data can betransmitted through a network.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) computer readable medium (or software) stored on anelectronic storage location of the server 701, such as, for example, onthe memory 710, or electronic storage unit 715. During use, the code canbe executed by the processor 705. In some cases, the code can beretrieved from the storage unit 715 and stored on the memory 710 forready access by the processor 705. In some situations, the electronicstorage unit 715 can be precluded, and machine-executable instructionsare stored on memory 710. Alternatively, the code can be executed on asecond computer system 740.

Aspects of the systems and methods provided herein, such as the server701, can be embodied in programming. Various aspects of the technologymay be thought of as “products” or “articles of manufacture” typicallyin the form of machine (or processor) executable code and/or associateddata that is carried on or embodied in a type of machine readable medium(e.g., computer readable medium). Machine-executable code can be storedon an electronic storage unit, such memory (e.g., read-only memory,random-access memory, flash memory) or a hard disk. “Storage” type mediacan include any or all of the tangible memory of the computers,processors or the like, or associated modules thereof, such as varioussemiconductor memories, tape drives, disk drives and the like, which mayprovide non-transitory storage at any time for the software programming.All or portions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical, and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless likes, opticallinks, or the like, also may be considered as media bearing thesoftware. As used herein, unless restricted to non-transitory, tangible“storage” media, terms such as computer or machine “readable medium”refer to any medium that participates in providing instructions to aprocessor for execution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, tangible storage medium,a carrier wave medium, or physical transmission medium. Non-volatilestorage media can include, for example, optical or magnetic disks, suchas any of the storage devices in any computer(s) or the like, such maybe used to implement the system. Tangible transmission media caninclude: coaxial cables, copper wires, and fiber optics (including thewires that comprise a bus within a computer system). Carrier-wavetransmission media may take the form of electric or electromagneticsignals, or acoustic or light waves such as those generated during radiofrequency (RF) and infrared (IR) data communications. Common forms ofcomputer-readable media therefore include, for example: a floppy disk, aflexible disk, hard disk, magnetic tape, any other magnetic medium, aCD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame,any other physical storage medium with patterns of holes, a RAM, a ROM,a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, acarrier wave transporting data or instructions, cables, or linkstransporting such carrier wave, or any other medium from which acomputer may read programming code and/or data. Many of these forms ofcomputer readable media may be involved in carrying one or moresequences of one or more instructions to a processor for execution.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES Example 1 Media for Growing Bacteria Strains

A microbial strain of the invention can be grown using the mediadescribed in this example.

For preparing the media, combine all ingredients shown in Table 7:

TABLE 7 Recipe for growth media PYGveg Component Amount per literGlucose 5 g K₂HPO₄ 2 g Tween 80 1 ml Cystein-HCl 0.5 g Yeast extract 10g HiVeg Extract 5 g HiVeg Peptone #1 5 g HiVeg Peptone #3 5 g VitaminMix 100x 10 ml Salt solution 40 ml Salt solution Component grams perliter CaCl₂ 2H₂O 0.02 MgSO₄ 7H₂O 0.02 K₂HPO₄ 0.04 KH₂PO₄ 0.04 NaHCO₃ 0.4NaCl 0.08

Dissolve the ingredients in boiling water, which can contain lessoxygen. Purge with nitrogen gas until the medium is completelyanaerobic. Seal bottle with rubber septum. Let the medium cool down.Perform aliquoting of the anaerobic medium in a glove box to maintainanaerobic condition. Autoclave the medium for about 20 minutes at 121degrees Celsius. Let the medium cool down and add the appropriate amountof 100× vitamins, shown in Table 8 below, to result in 1× final solutionof growth medium.

TABLE 8 Vitamin solution Vitamin Solution milligrams per Component literD-biotin 0.2 Ca-pantothenate 2.5 myoinositol 20 p-aminobenzoic acid 0.5pyridoxine 5 hydrochloride riboflavine 0.5 thiamine dichloride 10vitamin B12 0.2 nicotinic acid 5

FIG. 14 illustrates exemplary data for short chain fatty acidquantification in different media (e.g., RCM, PYG) by strain. The shortchain fatty acid quantification shows that the predicted genomicfunction of the strains matches the actual function. This can be similarfor different media. In one non-limiting example, strain 1 can beBifidobacterium adolescentis (BADO). In one non-limiting example, strain2 can be Bifidobacterium infantis (BINF). In one non-limiting example,strain 3 can be Bifidobacterium longum (BLON). In one non-limitingexample, strain 4 can be Clostridium beijerinckii (CBEI). In onenon-limiting example, strain 5 can be Clostridium butyricum (CBUT). Inone non-limiting example, strain 6 can be Clostridium indolis (CIND). Inone non-limiting example, strain 7 can be Eubacterium hallii (EHAL).

FIG. 15 illustrates that improved media of the invention (e.g.,PYGveg+vit+salt+buffer) can result in higher peak bacterial density. Inone non-limiting example, strain 1 can be Akkermansia muciniphila(AMUC). In one non-limiting example, strain 2 can be CBEI. In onenon-limiting example, strain 3 can be EHAL. In one non-limiting example,strain 4 can be CIND. In one non-limiting example, strain 5 can be BLON.In one non-limiting example, strain 6 can be BADO. In one non-limitingexample, strain 7 can be CBUT. In one non-limiting example, strain 8 canbe BINF.

Example 2 Stability of Strains in Formulation

FIG. 9 illustrates the stability of microbial strains Clostridiumbutyricum (CBUT), Clostridium beijerinckii (CBEI), Bifidobacteriumlongum (BLON), and Bifidobacterium infantis (BINF) when presentindividually or alone as compared to that observed when present togetherin a formulation, for example, WB0002 and WB0003.

In one non-limiting example of the invention, WB0002 comprisesClostridium butyricum (CBUT), Clostridium beijerinckii (CBEI),Bifidobacterium longum (BLON), and Bifidobacterium infantis (BINF), B.adolescentis, A. muciniphila, E. hallii, and C. indolis.

In one non-limiting example of the invention, WB0003 comprises thestrains Clostridium butyricum (CBUT), Clostridium beijerinckii (CBEI),Bifidobacterium longum (BLON), Bifidobacterium infantis (BINF), B.adolescentis, A. muciniphila, E. hallii, and C. indolis, and a prebiotic(e.g. inulin).

Example 3 Treatment of a Metabolic Condition with a MicrobialComposition

FIGS. 10 and 11A and B illustrate results of a preclinical study testingthe effect of a microbial composition of the invention (e.g., WB0002 andWB0003) on diet-induced obese mice.

In one non-limiting example of the invention, WB0002 comprisesClostridium butyricum (CBUT), Clostridium beijerinckii (CBEI),Bifidobacterium longum (BLON), Bifidobacterium infantis (BINF), B.adolescentis, A. muciniphila, E. hallii, and C. indolis.

In one non-limiting example of the invention, WB0003 comprises thestrains Clostridium butyricum (CBUT), Clostridium beijerinckii (CBEI),Bifidobacterium longum (BLON), Bifidobacterium infantis (BINF), B.adolescentis, A. muciniphila, E. hallii, and C. indolis, and a prebiotic(e.g. inulin).

As illustrated in FIG. 10, the microbial composition WB00003 resulted inincreased weight loss during the dosing period.

FIGS. 11A and B illustrate glucose control in mice dosed withformulations of the invention as compared with controls.

Example 4 Treatment of a Metabolic Condition with a MicrobialComposition

A subject with a metabolic condition, for example, obesity, insulininsensitivity, T2DM, and/or T1DM comes to a medical professional fortreatment.

The medical professional prescribes a microbial-based oral compositioncomprising the microbial strains Akkermansia muciniphila,Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacteriumlongum, Clostridium beijerinckii, Clostridium butyricum, Clostridiumindolis, and Eubacterium hallii. The composition may additionallycomprise Faecalibacterium prausnitzii in some embodiments. Each strainis present in a range of about 10{circumflex over ( )}8 to about10{circumflex over ( )}9 CFU in the composition. The compositionadditionally comprises inulin at a concentration of about 70 mg/mL. Theexpected delivery form of the oral composition is an enteric-coated(e.g., pH sensitive polymer Eudragit FS30D) pill that can protectagainst stomach acidity and deliver to the ileum/upper colon region ofthe subject. The enteric coating is designed to dissolve at a pH greaterthan about 6.5-7. In some embodiments, the oral composition isadministered as a liquid capsule.

The subject is administered the composition before food intake (e.g., 1hour before meals) twice daily for fourteen consecutive days. In somecases, the composition is administered simultaneously with food intake.

The microbial composition alters the microbial habitat of the gut of thesubject to that of a healthy subject. The subject loses weight. Thesubject's metabolic condition, for example, obesity, insulininsensitivity, T2DM, and/or T1DM is treated by the composition.

In some embodiments, a sample is taken from the subject to personalizethe composition of the microbial-based oral composition. For example, ifthe subject has a low level of one or more of the microbial strains, amicrobial-based oral composition may be administered that contains theone or more of the microbial strains that the subject is deficient in.

Example 5 Study to Evaluate Microbial Compositions in Treating aMetabolic Condition

Objective: The purpose of the study is to assess the effect of microbialcompositions of the invention in treating a metabolic condition, forexample, obesity, insulin insensitivity, T2DM, and/or T1DM.

Methods: Twenty subjects with a metabolic disorder, enter adouble-blind, placebo controlled and randomized study.

-   -   1) Experimental group: Ten subjects are given oral compositions        containing the active composition comprising: Akkermansia        muciniphila, Bifidobacterium adolescentis, Bifidobacterium        infantis, Bifidobacterium longum, Clostridium beijerinckii,        Clostridium butyricum, Clostridium indolis, and Eubacterium        hallii strains, and the prebiotic inulin. The composition can        additionally comprise Faecalibacterium prausnitzii. The        composition is taken once a day for 3 weeks before or        simultaneously with meals. Parameters observed are weight of the        subject and glucose tolerance before and after administration of        the composition daily for 3 weeks.    -   2) Control group: Ten subjects are given a placebo pill. The        placebo is taken once a day for 3 weeks. Parameters observed are        weight of the subject and glucose tolerance before and after        administration of the composition daily for 3 weeks.

Predicted Results: Following treatment, subjects in the experimentalgroup have a restored gut microbiome, reduction in weight in obesesubjects, and increased glucose tolerance compared with the controlgroup.

Example 6 Treatment of a Metabolic Condition with a MicrobialComposition

A subject with a metabolic condition, for example, obesity, insulininsensitivity, T2DM, and/or T1DM comes to a medical professional fortreatment.

The medical professional prescribes a microbial-based oral compositioncomprising the microbial strains Clostridium butyricum, Clostridiumbeijerinckii, Bifidobacterium longum, and Bifidobacterium infantis. Eachstrain is present in a range of about 10{circumflex over ( )}7 to about10{circumflex over ( )}12 CFU in the composition. The compositionadditionally comprises a prebiotic at a concentration of about 70 mg/mL.The expected delivery form of the oral composition is an enteric-coated(e.g., pH sensitive polymer Eudragit FS30D) pill that can protectagainst stomach acidity and deliver to the ileum/upper colon region ofthe subject. The enteric coating is designed to dissolve at a pH greaterthan about 6.5-7. In some embodiments, the oral composition isadministered as a liquid capsule.

The subject is administered the composition before food intake (e.g., 1hour before meals) twice daily for fourteen consecutive days.

The microbial composition alters the microbial habitat of the gut of thesubject to that of a healthy subject. The subject loses weight. Thesubject's metabolic condition, for example, obesity, insulininsensitivity, T2DM, and/or T1DM is treated by the composition.

Example 7 Study to Evaluate Microbial Compositions in Treating aMetabolic Condition

Objective: The purpose of the study is to assess the effect of microbialcompositions of the invention in treating a metabolic condition, forexample, obesity, insulin insensitivity, T2DM, and/or T1DM.

Methods: Twenty subjects with a metabolic disorder, enter adouble-blind, placebo controlled and randomized study.

-   -   3) Experimental group: Ten subjects are given oral compositions        containing the active composition comprising: Clostridium        butyricum, Clostridium beijerinckii, Bifidobacterium longum, and        Bifidobacterium infantis. The composition is taken once a day        for 3 weeks before meals. Parameters observed are weight of the        subject and glucose tolerance before and after administration of        the composition daily for 3 weeks.    -   4) Control group: Ten subjects are given a placebo pill. The        placebo is taken once a day for 3 weeks. Parameters observed are        weight of the subject and glucose tolerance before and after        administration of the composition daily for 3 weeks.

Predicted Results: Following treatment, subjects in the experimentalgroup have a restored gut microbiome, reduction in weight (e.g., inobese subjects), and increased glucose tolerance compared with thecontrol group.

What is claimed is:
 1. A method of producing a microbial compositionsuitable for human consumption, the method comprising: (a) culturingAkkermansia muciniphila in a culture medium comprising substantially aplant-based culture medium, thereby generating cultured Akkermansiamuciniphila; and (b) formulating the cultured Akkermansia muciniphilainto a unit dosage form, wherein the unit dosage form comprises: (i) thecultured Akkermansia muciniphila; and (ii) a second microbe comprising a16S rRNA sequence that comprises at least about 97% sequence identity toa 16S rRNA sequence of Eubacterium hallii, thereby producing themicrobial composition suitable for human consumption.
 2. The method ofclaim 1, wherein the unit dosage form comprises the cultured Akkermansiamuciniphila and the second microbe in a powdered form.
 3. The method ofclaim 1, wherein the unit dosage form comprises the cultured Akkermansiamuciniphila and the second microbe in a lyophilized form.
 4. The methodof claim 3, wherein the culturing of the Akkermansia muciniphila iscarried out anaerobically.
 5. The method of claim 1, wherein the unitdosage form is a capsule.
 6. The method of claim 1, wherein the unitdosage form is a food composition in a form of a bar.
 7. The method ofclaim 1, wherein the unit dosage form further comprises the plant-basedculture medium.
 8. The method of claim 7, wherein the unit dosage formcomprises the plant-based culture medium in a powdered form.
 9. Themethod of claim 1, wherein the unit dosage form comprises at least 1×10⁵CFUs of the cultured Akkermansia muciniphila.
 10. The method of claim 1,wherein the unit dosage form further comprises one or more of:Clostridium beijerinckii, Clostridium butyricum, Clostridium indolis,Bifidobacterium infantis, or Faecalibacterium prausnitzii.
 11. Themethod of claim 1, wherein the second microbe is Eubacterium hallii. 12.The method of claim 1, wherein the unit dosage form comprises at least1×10⁵ CFUs of the second microbe.
 13. The method of claim 1, wherein theunit dosage form does not contain dairy.
 14. The method of claim 1,wherein the unit dosage form further comprises Bifidobacterium infantis.15. The method of claim 1, wherein the plant-based culture mediumcomprises an amino acid source that is a plant-protein hydrolysate. 16.The method of claim 1, wherein the unit dosage form further comprises aprebiotic.
 17. The method of claim 1, wherein the culture medium furthercomprises one or more vitamins, one or more salts, a buffer, or acombination thereof.
 18. The method of claim 1, wherein the culturemedium consists essentially of the plant-based culture medium.
 19. Themethod of claim 1, wherein the culture medium consists of theplant-based culture medium, one or more salts, one or more vitamins, anda buffer.